Wnt antagonists and their use in the diagnosis and treatment of wnt-mediated disorders

ABSTRACT

The present invention provides for chimeric Wnt antagonists comprising a Frz domain component derived from a Frizzled protein, a secreted Frizzled related protein or Ror protein and an Fc immunoglobulin component, and their use in the treatment and diagnostic detection of cellular Wnt signaling and Wnt-mediated disorders, including cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/367,760, filed on Feb. 7, 2012, now abandoned, which is acontinuation of U.S. application Ser. No. 12/826,194, filed on Jun. 29,2010, now abandoned, which is a continuation of U.S. application Ser.No. 11/851,596, filed Sep. 7, 2007, now U.S. Pat. No. 7,947,277, whichin turn claims priority to U.S. Provisional Application No. 60/825,063,filed Sep. 8, 2006, and U.S. Provisional Application No. 60/951,175,filed Jul. 20, 2007, the contents of which are incorporated by referencein their entirety herein.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Dec. 6, 2016, is namedP02368-US-6SeqListing.txt and is 240,326 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to the regulation of cellgrowth. More specifically, the present invention relates to inhibitorsof the Wnt pathway as well as to their use in the diagnosis andtreatment of disorders characterized by the activation of Wnt pathwaysignaling, as well as to the modulation of cellular events mediated byWnt pathway signaling.

BACKGROUND OF THE INVENTION

The Wnt signaling pathway's association with carcinogenesis began as aresult of early observations and experiments in certain murine mammarytumors. Wnt-1 proto-oncogene (Int-1) was originally identified frommammary tumors induced by mouse mammary tumor virus (MMTV) due to aninsertion of a viral DNA sequence. Nusse et al., Cell 1982; 31: 99-109.The result of such viral integration was unregulated expression of Int-1resulting in the formation of tumors. Vanooyen, A. et al., Cell 1984;39: 233-240; Nusse, R. et al., Nature 1984; 307: 131-136; Tsukamoto etal., Cell 1988; 55: 619-625. Subsequent sequence analysis demonstratedthat the Int-1 was a mammalian homolog of the Drosophila gene Wingless(Wg), which was implicated in development, and the terms were thencombined to create “Wnt” to identify this family of proteins.

The human Wnt gene family of secreted ligands has now grown to at least19 members (e.g., Wnt-1 (RefSeq.: NM_005430), Wnt-2 (RefSeq.:NM_003391), Wnt-2B (Wnt-13) (RefSeq.: NM 004185), Wnt-3 (ReSeq.:NM_030753), Wnt3a (RefSeq.: NM_033131), Wnt-4 (RefSeq.: NM_030761),Wnt-5A (RefSeq.: NM_003392), Wnt-5B (RefSeq.: NM_032642), Wnt-6(RefSeq.: NM_006522), Wnt-7A (RefSeq.: NM_004625), Wnt-7B (RefSeq.:NM_058238), Wnt-8A (RefSeq.: NM_058244), Wnt-8B (RefSeq.: NM_003393),Wnt-9A (Wnt-14) (RefSeq.: NM_003395), Wnt-9B (Wnt-15) (RefSeq.:NM_003396), Wnt-10A (RefSeq.: NM_025216), Wnt-10B (RefSeq.: NM_003394),Wnt-11 (RefSeq.: NM_004626), Wnt-16 (RefSeq.: NM_016087)). Each memberhas varying degrees of sequence identity but all contain 23-24 conservedcysteine residues which show highly conserved spacing. McMahon, A P etal., Trends Genet. 1992; 8: 236-242; Miller, J R. Genome Biol. 2002;3(1): 3001.1-3001.15. The Wnt proteins are small (i.e., 39-46 kD)acylated, secreted glycoproteins which play key roles in bothembryogenesis and mature tissues. During embryological development, theexpression of Wnt proteins is important in patterning through control ofcell proliferation and determination of stem cell fate. The Wntmolecules are also palmitoylated, and thus are more hydrophobic thanwould be otherwise predicted by analysis of the amino acid sequencealone. Willert, K. et al., Nature 2003; 423: 448-52. The site or sitesof palmitoylation are also believed to be essential for function.

The Wnt proteins act as ligands to activate the Frizzled (Frz) family ofseven-pass transmembrane receptors. Ingham, P. W. Trends Genet. 1996;12: 382-384; YangSnyder, J. et al., Curr. Biol. 1996; 6: 1302-1306;Bhanot, P. et al., Nature 1996; 382: 225-230. There are ten knownmembers of the Frz family (e.g., Frz1, Frz2, Frz3 . . . Frz10), eachcharacterized by the presence of a cysteine rich domain (CRD). Huang etal., Genome Biol. 2004; 5: 234.1-234.8. There is a great degree ofpromiscuity between the various Wnt-Frizzled interactions, but Wnt-Frzbinding must also incorporate the LDL receptor related proteins (LRP5 orLRP6) and the membrane and the cytoplasmic protein Dishevelled (Dsh) toform an active signaling complex.

The binding of Wnt to Frizzled can activate signaling via either thecanonical Wnt signaling pathway, thereby resulting in stabilization andincreased transcriptional activity of β-catenin [Peifer, M. et al.,Development 1994; 120: 369-380; Papkoff, J. et al., Mol. Cell Biol.1996; 16: 2128-2134] or non-canonical signaling, such as through theWnt/planar cell polarity (Wnt/PCP) or Wnt-calcium (Wnt/Ca²⁺) pathway.Veeman, M. T. et al., Dev. Cell 2003; 5: 367-377.

The canonical Wnt signaling pathway is the most relevant of the Wntsignaling pathways to the development of cancer. Ilyas, M. J. Pathol.2005; 205: 130-144. Normal activation of this pathway begins a series ofdownstream events culminating in the stabilization and increased levelsof the protein β-catenin. This protein is normally an inactivecytoplasmic protein, and is found at the cell membrane bound to proteinsincluding e-cadherin. In the absence of Wnt ligand, phosphorylatedcytoplasmic β-catenin is normally rapidly degraded. Upon activation ofthe canonical pathway, unphosphorylated β-catenin is transported to thenucleus where it further results in transcriptional activation ofvarious target genes. The subsequent upregulation in transcription ofthese target genes leads to changes in the cell, and continuous,unregulated expression of such target genes results in tumordevelopment. Since aberrant Wnt signaling appears to be a necessaryprecursor in carcinogenesis, effective inhibitors of Wnt signaling areof great interest as cancer therapeutics.

The use of soluble receptors as antagonists to ligand-receptorinteractions is known in the art. Such molecules can be effectivetherapeutic antagonists if they bind the free ligand in a manner so asto prevent the initial receptor activation step of the signalingpathway. Soluble minimal extracellular domain (ECD) fragments of thecysteine-rich domain (CRD) of a Frizzled receptor which exhibit bindingto Wnt have been identified, based on crystallography data. Dann et al.,Nature 412: 86-90 (2001). However, while such Frizzled fragments didexhibit binding to Wnt ligand, such fragments are unsuitable fortherapeutics because of their rapid degradation in vivo.

The use of a soluble Frizzled domain coupled to an immunoglobulin Fc asa potential Wnt antagonist has been proposed. Therapeutic Opportunitiesof the Wnt Signaling Pathway in Cancer, New York Academy of Sciences,Oct. 25, 2005; Hsieh, J-C. et al., PNAS, 96: 3546-3551 (1999). However,prior to the present invention, attempts at creating a soluble Frizzledreceptor-Fc fusion therapeutic were not successful. For example, onesuch chimera based on residues 1-173 of the Frz8 CRD (Frz (173)-Fc, SEQID NO: 113) had suboptimal efficacy (FIG. 12), and was unstable in vivo(FIG. 11). Moreover, the Frz (173)-Fc chimera only reduced the rate ofincrease in tumor volume (as opposed to shrinking starting tumorvolume). Additionally, while the creation of Fc fusions is generallyknown as one technique to improve the in vivo stability of the resultingconstruct, the creation of effective therapeutic Fc constructs can bedifficult owing to a number of problems, including improper proteinfolding of the new protein construct and steric hindrance of the fusionconstruct to the target.

Thus, a need exits for a Wnt antagonist therapeutic with enhanced invivo stability that acts to inhibit Wnt ligand induced cellularsignaling.

SUMMARY OF THE INVENTION

The invention provides for compositions and their use in methods ofdiagnosing and treating Wnt-mediated disorder, such as cancer, and ininhibiting cellular Wnt signaling. Specifically, the invention providesfor Wnt antagonists that are chimeric molecules comprising a Frizzleddomain component, such as a polypeptide derived from a Frizzled (Frz)protein, a Frizzled related protein (sFRP) or another protein (e.g.,Ror-1, -2, etc.), and an immunoglobulin Fc domain, and their use inmethods of diagnosing and treating Wnt-mediated disorders and ininhibiting cellular Wnt signaling.

One aspect of the invention provides for a Wnt antagonist comprising aFrizzled domain component and a Fc domain. The Frizzled domain componentof the Wnt antagonist comprises a polypeptide derived from a Frzprotein, a FRP protein, or a Ror protein. In one embodiment, the Wntantagonist is active in vivo for at least 1 hour. In another embodiment,the Wnt antagonist is active in vivo for at least 5 hours. In anotherembodiment, the Wnt antagonist has an in vivo half-life of at least 1day. In yet another embodiment, the Wnt antagonist has an in vivohalf-life of at least 2 days.

In a further embodiment, the Frizzled domain component comprises aminimal CRD (ECD) domain from a Frz polypeptide selected from the groupconsisting of hFrz1 (SEQ ID NO: 18), hFrz2 (SEQ ID NO: 19), hFrz3 (SEQID NO: 20), hFrz4 (SEQ ID NO: 21), hFrz5 (SEQ ID NO: 22), hFrz6 (SEQ IDNO: 23), hFrz7 (SEQ ID NO: 24), hFrz8 (SEQ ID NO: 25), hFrz9 (SEQ ID NO:26), and hFrz10 (SEQ ID NO: 27), and active variants thereof. In yet afurther embodiment, the Frizzled domain component comprises a minimalCRD (ECD) domain from a sFRP polypeptide selected from the groupconsisting of sFRP1 (SEQ ID NO: 28), sFRP2 (SEQ ID NO: 29), sFRP3 (SEQID NO: 30), sFRP4 (SEQ ID NO: 31), and sFRP5 (SEQ ID NO: 32), and activevariants thereof. In yet a further embodiment, the Frizzled domaincomponent comprises a minimal CRD (ECD) domain from a Ror polypeptideselected from the group consisting of hRor1 (SEQ ID NO: 33), and hRor2(SEQ ID NO: 34), and active variants thereof.

In yet a further embodiment, the Frizzled domain component comprises amature Frz polypeptide selected from the group consisting of hFrz1 (SEQID NO: 50), hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ ID NO: 52), hFrz4 (SEQ IDNO: 53), hFrz5 (SEQ ID NO: 54), hFrz6 (SEQ ID NO: 55), hFrz7 (SEQ ID NO:56), hFrz8 (SEQ ID NO: 57), hFrz9 (SEQ ID NO: 58), and hFrz10 (SEQ IDNO: 59), and active variants thereof, or a mature sFrp polypeptideselected from the group consisting of sFRP1 (SEQ ID NO: 60), sFRP2 (SEQID NO: 61), sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), and sFRP5 (SEQID NO: 64), and active variants thereof, or a mature Ror polypeptideselected from the group consisting of hRor1 (SEQ ID NO: 65), and hRor2(SEQ ID NO: 66), and active variants thereof.

In a still further embodiment, the Frizzled domain component comprises apro-Frz polypeptide selected from the group consisting of hFrz1 (SEQ IDNO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID NO: 37), hFrz4 (SEQ ID NO:38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ ID NO: 40), hFrz7 (SEQ ID NO:41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ ID NO: 43), and hFrz10 (SEQ IDNO: 44), and active variants thereof, or a pro-sFrp polypeptide selectedfrom the group consisting of sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO:46), sFRP3 (SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO:49), and active variants thereof.

In one embodiment, the Wnt antagonist comprises a Fc component derivedfrom an immunoglobulin selected from the group consisting of IgG1, IgG2,IgG3 and IgG4. In another embodiment, the Fc is derived from an IgG1immunoglobulin. In yet another embodiment the Fc sequence comprises theFc shown in SEQ ID NO: 67 or SEQ ID NO: 68.

In one embodiment, the Wnt antagonist further comprises a linkerconnecting the Frizzled domain component to the Fc domain. In one suchembodiment, the linker is a peptide linker such as ESGGGGVT (SEQ ID NO:69), LESGGGGVT (SEQ ID NO: 70), GRAQVT (SEQ ID NO: 71), WRAQVT (SEQ IDNO: 72), and ARGRAQVT (SEQ ID NO: 73).

In particular embodiments, the Wnt antagonist comprises a polypeptideselected from the group consisting of in Frz8-Fc (SEQ ID NO: 74),Frz5-Fc (SEQ ID NO: 75), Frz1-Fc (SEQ ID NO: 76), Frz2-Fc (SEQ ID NO:77), Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ ID NO: 79), Frz6-Fc (SEQ IDNO: 80), Frz7-Fc (SEQ ID NO: 81), Frz9-Fc (SEQ ID NO: 82), Frz10-Fc (SEQID NO: 83), sFRP1-Fc (SEQ ID NO: 84), sFRP2 (SEQ ID NO: 85), sFRP3-Fc(SEQ ID NO: 86), sFRP4-Fc (SEQ ID NO: 87), and sFRP5-Fc (SEQ ID NO: 88).

Another aspect of the invention provides for a composition comprising atleast one pharmaceutically acceptable carrier or excipient and a Wntantagonist as described above.

Yet another aspect of the invention provides for a nucleic acid sequenceencoding any of the Wnt antagonists described above. In one embodiment,the nucleic acid encoding a Wnt antagonist further comprises a vectorcontaining control sequences to which the nucleic acid is operablylinked. In another embodiment, the vector is contained in host cells,such as a mammalian, insect, E. coli or yeast cell.

Another aspect of the invention provides for an article of manufacturecomprising a composition comprising at least one pharmaceuticallyacceptable carrier or excipient and a Wnt antagonist as described aboveand a container, wherein the Wnt antagonist is contained within thecontainer and the container further comprises (a) a label affixed to thecontainer, or (b) a package insert inside the container referring to theuse of the Wnt antagonist indicating use of the composition for thetherapeutic treatment or diagnostic detection of a Wnt-mediateddisorder.

Yet another aspect of the invention provides for a method of inhibitingWnt signaling in a cell comprising contacting the cell with an effectiveamount of a Wnt antagonist comprising a Frizzled domain component and aFc domain. The Frizzled domain component of the Wnt antagonist comprisesa polypeptide derived from a Frz protein, a FRP protein, or a Rorprotein. In one embodiment, the Wnt antagonist is active in vivo for atleast 1 hour. In another embodiment, the Wnt antagonist is active invivo for at least 5 hours. In another embodiment, the Wnt antagonist hasan in vivo half-life of at least 1 day. In yet another embodiment, theWnt antagonist has an in vivo half-life of at least 2 days.

In a further embodiment of this aspect, the Frizzled domain componentcomprises a minimal CRD (ECD) domain from a Frz polypeptide selectedfrom the group consisting of hFrz1 (SEQ ID NO: 18), hFrz2 (SEQ ID NO:19), hFrz3 (SEQ ID NO: 20), hFrz4 (SEQ ID NO: 21), hFrz5 (SEQ ID NO:22), hFrz6 (SEQ ID NO: 23), hFrz7 (SEQ ID NO: 24), hFrz8 (SEQ ID NO:25), hFrz9 (SEQ ID NO: 26), and hFrz10 (SEQ ID NO: 27), and activevariants thereof. In yet a further embodiment, the Frizzled domaincomponent comprises a minimal CRD (ECD) domain from a sFRP polypeptideselected from the group consisting of sFRP1 (SEQ ID NO: 28), sFRP2 (SEQID NO: 29), sFRP3 (SEQ ID NO: 30), sFRP4 (SEQ ID NO: 31), and sFRP5 (SEQID NO: 32), and active variants thereof. In yet a further embodiment,the Frizzled domain component comprises a minimal CRD (ECD) domain froma Ror polypeptide selected from the group consisting of hRor1 (SEQ IDNO: 33), and hRor2 (SEQ ID NO: 34), and active variants thereof.

In yet a further embodiment, the Frizzled domain component comprises amature Frz polypeptide selected from the group consisting of: (SEQ IDNO: 50), hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ ID NO: 52), hFrz4 (SEQ ID NO:53), hFrz5 (SEQ ID NO: 54), hFrz6 (SEQ ID NO: 55), hFrz7 (SEQ ID NO:56), hFrz8 (SEQ ID NO: 57), hFrz9 (SEQ ID NO: 58), and hFrz10 (SEQ IDNO: 59), and active variants thereof, or a mature sFrp polypeptideselected from the group consisting of sFRP1 (SEQ ID NO: 60), sFRP2 (SEQID NO: 61), sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), and sFRP5 (SEQID NO: 64), and active variants thereof, or a mature Ror polypeptideselected from the group consisting of hRor1 (SEQ ID NO: 65), and hRor2(SEQ ID NO: 66), and active variants thereof.

In a still further embodiment, the Frizzled domain component comprises apro-Frz polypeptide selected from the group consisting of hFrz1 (SEQ IDNO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID NO: 37), hFrz4 (SEQ ID NO:38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ ID NO: 40), hFrz7 (SEQ ID NO:41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ ID NO: 43), and hFrz10 (SEQ IDNO: 44), and active variants thereof, or a pro-sFrp polypeptide selectedfrom the group consisting of sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO:46), sFRP3 (SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO:49), and active variants thereof.

In one embodiment, the Wnt antagonist comprises a Fc component derivedfrom an immunoglobulin selected from the group consisting of IgG1, IgG2,IgG3 and IgG4. In another embodiment, the Fc is derived from an IgG1immunoglobulin. In yet another embodiment the Fc sequence shown in SEQID NO: 67 or SEQ ID NO: 68.

In one embodiment, Wnt antagonist further comprises a linker connectingthe Frizzled domain component to the Fc domain. In one embodiment, thelinker is a peptide linker such as ESGGGGVT (SEQ ID NO: 69), LESGGGGVT(SEQ ID NO: 70), GRAQVT (SEQ ID NO: 71), WRAQVT (SEQ ID NO: 72), andARGRAQVT (SEQ ID NO: 73).

In particular embodiments, the Wnt antagonist comprises a polypeptideselected from the group consisting of Frz8-Fc (SEQ ID NO: 74), Frz5-Fc(SEQ ID NO: 75), Frz1-Fc (SEQ ID NO: 76), Frz2-Fc (SEQ ID NO: 77),Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ ID NO: 79), Frz6-Fc (SEQ ID NO:80), Frz7-Fc (SEQ ID NO: 81), Frz9-Fc (SEQ ID NO: 82), Frz10-Fc (SEQ IDNO: 83), sFRP1-Fc (SEQ ID NO: 84), sFRP2 (SEQ ID NO: 85), sFRP3-Fc (SEQID NO: 86), sFRP4-Fc (SEQ ID NO: 87), and sFRP5-Fc (SEQ ID NO: 88).

In one embodiment of this method, the cell is contained within a mammaland the amount administered is a therapeutically effective amount. Inanother embodiment, the Wnt signaling results from activation of a Wntsignaling component through somatic mutation. In another embodiment, theinhibition of Wnt signaling results in the inhibition of growth of thecell. In yet another embodiment, the cell is a cancer cell.

Another aspect of the invention provides for a method of treating aWnt-mediated disorder in a mammal suffering therefrom, comprisingadministering to the mammal a therapeutically effective amount of a Wntantagonist comprising a Frizzled domain component and a Fc domain. TheFrizzled domain component of the Wnt antagonist comprises a polypeptidederived from a Frz protein, a FRP protein, or a Ror protein. In oneembodiment, the Wnt antagonist is active in vivo for at least 1 hour. Inanother embodiment, the Wnt antagonist is active in vivo for at least 5hours. In another embodiment, the Wnt antagonist has an in vivohalf-life of at least 1 day. In yet another embodiment, the Wntantagonist has an in vivo half-life of at least 2 days.

In a further embodiment of this aspect, the Frizzled domain componentcomprises a minimal CRD (ECD) domain from a Frz polypeptide selectedfrom the group consisting of hFrz1 (SEQ ID NO: 18), hFrz2 (SEQ ID NO:19), hFrz3 (SEQ ID NO: 20), hFrz4 (SEQ ID NO: 21), hFrz5 (SEQ ID NO:22), hFrz6 (SEQ ID NO: 23), hFrz7 (SEQ ID NO: 24), hFrz8 (SEQ ID NO:25), hFrz9 (SEQ ID NO: 26), and hFrz10 (SEQ ID NO: 27), and activevariants thereof. In yet a further embodiment, the Frizzled domaincomponent comprises a minimal CRD (ECD) domain from a sFRP polypeptideselected from the group consisting of sFRP1 (SEQ ID NO: 28), sFRP2 (SEQID NO: 29), sFRP3 (SEQ ID NO: 30), sFRP4 (SEQ ID NO: 31), and sFRP5 (SEQID NO: 32), and active variants thereof. In yet a further embodiment,the Frizzled domain component comprises a minimal CRD (ECD) domain froma Ror polypeptide selected from the group consisting of hRor1 (SEQ IDNO: 33), and hRor2 (SEQ ID NO: 34), and active variants thereof.

In yet a further embodiment, the Frizzled domain component comprises amature Frz polypeptide selected from the group consisting of: (SEQ IDNO: 50), hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ ID NO: 52), hFrz4 (SEQ ID NO:53), hFrz5 (SEQ ID NO: 54), hFrz6 (SEQ ID NO: 55), hFrz7 (SEQ ID NO:56), hFrz8 (SEQ ID NO: 57), hFrz9 (SEQ ID NO: 58), and hFrz10 (SEQ IDNO: 59), and active variants thereof, or a mature sFrp polypeptideselected from the group consisting of sFRP1 (SEQ ID NO: 60), sFRP2 (SEQID NO: 61), sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), and sFRP5 (SEQID NO: 64), and active variants thereof, or a mature Ror polypeptideselected from the group consisting of hRor1 (SEQ ID NO: 65), and hRor2(SEQ ID NO: 66), and active variants thereof.

In still further embodiments, the Frizzled domain component comprises apro-Frz polypeptide selected from the group consisting of hFrz1 (SEQ IDNO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID NO: 37), hFrz4 (SEQ ID NO:38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ ID NO: 40), hFrz7 (SEQ ID NO:41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ ID NO: 43), and hFrz10 (SEQ IDNO: 44), and active variants thereof, or a pro-sFrp polypeptide selectedfrom the group consisting of sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO:46), sFRP3 (SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO:49), and active variants thereof.

In one embodiment, the Wnt antagonist comprises a Fc component derivedfrom an immunoglobulin selected from the group consisting of IgG1, IgG2,IgG3 and IgG4. In another embodiment, the Fc is derived from an IgG1immunoglobulin. In yet another embodiment the Fc sequence shown in SEQID NO: 67 or SEQ ID NO: 68.

In one embodiment, Wnt antagonist further comprises a linker connectingthe Frizzled domain component to the Fc domain. In one embodiment, thelinker is a peptide linker such as ESGGGGVT (SEQ ID NO: 69), LESGGGGVT(SEQ ID NO: 70), GRAQVT (SEQ ID NO: 71), WRAQVT (SEQ ID NO: 72), andARGRAQ VT (SEQ ID NO: 73).

In particular embodiments, the Wnt antagonist comprises a polypeptideselected from the group consisting of Frz8-Fc (SEQ ID NO: 74), Frz5-Fc(SEQ ID NO: 75), Frz1-Fc (SEQ ID NO: 76), Frz2-Fc (SEQ ID NO: 77),Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ ID NO: 79), Frz6-Fc (SEQ ID NO:80), Frz7-Fc (SEQ ID NO: 81), Frz9-Fc (SEQ ID NO: 82), Frz10-Fc (SEQ IDNO: 83), sFRP1-Fc (SEQ ID NO: 84), sFRP2 (SEQ ID NO: 85), sFRP3-Fc (SEQID NO: 86), sFRP4-Fc (SEQ ID NO: 87), and sFRP5-Fc (SEQ ID NO: 88).

In one embodiment of this method, the disorder is a cell proliferativedisorder associated with aberrant Wnt signaling activity. In anotherembodiment, the aberrant Wnt signaling activity results from increasedexpression of a Wnt protein. In yet another embodiment, the cellproliferative disorder is cancer, such as of colon cancer, colorectalcancer, breast cancer, leukemia, gliomas, or medulloblastomas.

Yet another aspect of the invention provides for a method for detectingthe presence of a Wnt protein, comprising contacting the sample with aWnt antagonist as described above, where the presence of a complex, orthe binding level between the Wnt antagonist and Wnt protein isindicative of the presence of a Wnt protein and/or signaling. In oneembodiment, the method further comprises determining if the level of Wntsignaling is aberrant, the method further comprising comparing the levelof binding in the sample to the level in a second sample known to havephysiologically normal Wnt signaling. A level of binding in the samplethat is higher or lower than that of the second sample is indicative ofaberrant Wnt signaling. In yet another embodiment, the aberrant Wntsignaling is further indicative of the presence of a Wnt-mediateddisorder, such as cancer.

Another aspect of the invention provides for a method of modulating theexpression of a Wnt target gene in a cell characterized by activated orexcessive Wnt signaling, comprising contact the cell with an effectiveamount of a Wnt antagonist described above.

Yet another aspect of the invention provides for a method oftherapeutically treating a Wnt-mediated cancer, comprising administeringa therapeutically effective amount of a Wnt antagonist comprising aFrizzled domain component and a Fc domain. The Frizzled domain componentof the Wnt antagonist comprises a polypeptide derived from a Frzprotein, a FRP protein, or a Ror protein. In one embodiment, the Wntantagonist is active in vivo for at least 1 hour. In another embodiment,the Wnt antagonist is active in vivo for at least 5 hours. In anotherembodiment, the Wnt antagonist has an in vivo half-life of at least 1day. In yet another embodiment, the Wnt antagonist has an in vivohalf-life of at least 2 days.

In a further embodiment of this aspect, the Frizzled domain componentcomprises a minimal CRD (ECD) domain from a Frz polypeptide selectedfrom the group consisting of hFrz1 (SEQ ID NO: 18), hFrz2 (SEQ ID NO:19), hFrz3 (SEQ ID NO: 20), hFrz4 (SEQ ID NO: 21), hFrz5 (SEQ ID NO:22), hFrz6 (SEQ ID NO: 23), hFrz7 (SEQ ID NO: 24), hFrz8 (SEQ ID NO:25), hFrz9 (SEQ ID NO: 26), and hFrz10 (SEQ ID NO: 27), and activevariants thereof. In yet a further embodiment, the Frizzled domaincomponent comprises a minimal CRD (ECD) domain from a sFRP polypeptideselected from the group consisting of sFRP1 (SEQ ID NO: 28), sFRP2 (SEQID NO: 29), sFRP3 (SEQ ID NO: 30), sFRP4 (SEQ ID NO: 31), and sFRP5 (SEQID NO: 32), and active variants thereof. In yet a further embodiment,the Frizzled domain component comprises a minimal CRD (ECD) domain froma Ror polypeptide selected from the group consisting of hRor1 (SEQ IDNO: 33), and hRor2 (SEQ ID NO: 34), and active variants thereof.

In yet a further embodiment, the Frizzled domain component comprises amature Frz polypeptide selected from the group consisting of: (SEQ IDNO: 50), hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ ID NO: 52), hFrz4 (SEQ ID NO:53), hFrz5 (SEQ ID NO: 54), hFrz6 (SEQ ID NO: 55), hFrz7 (SEQ ID NO:56), hFrz8 (SEQ ID NO: 57), hFrz9 (SEQ ID NO: 58), and hFrz10 (SEQ IDNO: 59), and active variants thereof, or a mature sFrp polypeptideselected from the group consisting of sFRP1 (SEQ ID NO: 60), sFRP2 (SEQID NO: 61), sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), and sFRP5 (SEQID NO: 64), and active variants thereof, or a mature Ror polypeptideselected from the group consisting of hRor1 (SEQ ID NO: 65), and hRor2(SEQ ID NO: 66), and active variants thereof.

In a still further embodiment, the Frizzled domain component comprises apro-Frz polypeptide selected from the group consisting of hFrz1 (SEQ IDNO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID NO: 37), hFrz4 (SEQ ID NO:38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ ID NO: 40), hFrz7 (SEQ ID NO:41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ ID NO: 43), and hFrz10 (SEQ IDNO: 44), and active variants thereof, or a pro-sFrp polypeptide selectedfrom the group consisting of sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO:46), sFRP3 (SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO:49), and active variants thereof.

In one embodiment, the Wnt antagonist comprises a Fc component derivedfrom an immunoglobulin selected from the group consisting of IgG1, IgG2,IgG3 and IgG4. In another embodiment, the Fc is derived from an IgG1immunoglobulin. In yet another embodiment the Fc sequence shown in SEQID NO: 67 or SEQ ID NO: 68.

In one embodiment, Wnt antagonist further comprises a linker connectingthe Frizzled domain component to the Fc domain. In one embodiment, thelinker is a peptide linker such as ESGGGGVT (SEQ ID NO: 69), LESGGGGVT(SEQ ID NO: 70), GRAQVT (SEQ ID NO: 71), WRAQVT (SEQ ID NO: 72), andARGRAQVT (SEQ ID NO: 73).

In particular embodiments, the Wnt antagonist comprises a polypeptideselected from the group consisting of Frz8-Fc (SEQ ID NO: 74), Frz5-Fc(SEQ ID NO: 75), Frz1-Fc (SEQ ID NO: 76), Frz2-Fc (SEQ ID NO: 77),Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ ID NO: 79), Frz6-Fc (SEQ ID NO:80), Frz7-Fc (SEQ ID NO: 81), Frz9-Fc (SEQ ID NO: 82), Frz10-Fc (SEQ IDNO: 83), sFRP1-Fc (SEQ ID NO: 84), sFRP2 (SEQ ID NO: 85), sFRP3-Fc (SEQID NO: 86), sFRP4-Fc (SEQ ID NO: 87), and sFRP5-Fc (SEQ ID NO: 88).

The administration of the antagonist arrests any subsequent increase insize or advancement in severity of the cancer. In one embodiment, theadministration of the Wnt antagonist results in the reduction in size orseverity of the cancer. In another embodiment, the administration of theWnt antagonist reduces the tumor burden of the cancer. In yet anotherembodiment, the administration of the Wnt antagonist kills the cancer.

Another aspect of the invention provides for the use of a Wnt antagonistin the manufacture of a medicament for the treatment of a cellproliferative disorder. Wnt antagonist comprises a Frizzled domaincomponent and a Fc domain. The Frizzled domain component of the Wntantagonist comprises a polypeptide derived from a Frz protein, a FRPprotein, or a Ror protein. In one embodiment, the Wnt antagonist isactive in vivo for at least 1 hour. In another embodiment, the Wntantagonist is active in vivo for at least 5 hours. In anotherembodiment, the Wnt antagonist has an in vivo half-life of at least 1day. In yet another embodiment, the Wnt antagonist has an in vivohalf-life of at least 2 days.

In a further embodiment of this aspect, the Frizzled domain componentcomprises a minimal CRD (ECD) domain from a Frz polypeptide selectedfrom the group consisting of hFrz1 (SEQ ID NO: 18), hFrz2 (SEQ ID NO:19), hFrz3 (SEQ ID NO: 20), hFrz4 (SEQ ID NO: 21), hFrz5 (SEQ ID NO:22), hFrz6 (SEQ ID NO: 23), hFrz7 (SEQ ID NO: 24), hFrz8 (SEQ ID NO:25), hFrz9 (SEQ ID NO: 26), and hFrz10 (SEQ ID NO: 27), and activevariants thereof. In yet a further embodiment, the Frizzled domaincomponent comprises a minimal CRD (ECD) domain from a sFRP polypeptideselected from the group consisting of sFRP1 (SEQ ID NO: 28), sFRP2 (SEQID NO: 29), sFRP3 (SEQ ID NO: 30), sFRP4 (SEQ ID NO: 31), and sFRP5 (SEQID NO: 32), and active variants thereof. In yet a further embodiment,the Frizzled domain component comprises a minimal CRD (ECD) domain froma Ror polypeptide selected from the group consisting of hRor1 (SEQ IDNO: 33), and hRor2 (SEQ ID NO: 34), and active variants thereof.

In yet a further embodiment, the Frizzled domain component comprises amature Frz polypeptide selected from the group consisting of: (SEQ IDNO: 50), hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ ID NO: 52), hFrz4 (SEQ ID NO:53), hFrz5 (SEQ ID NO: 54), hFrz6 (SEQ ID NO: 55), hFrz7 (SEQ ID NO:56), hFrz8 (SEQ ID NO: 57), hFrz9 (SEQ ID NO: 58), and hFrz10 (SEQ IDNO: 59), and active variants thereof, or a mature sFrp polypeptideselected from the group consisting of sFRP1 (SEQ ID NO: 60), sFRP2 (SEQID NO: 61), sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), and sFRP5 (SEQID NO: 64), and active variants thereof, or a mature Ror polypeptideselected from the group consisting of hRor1 (SEQ ID NO: 65), and hRor2(SEQ ID NO: 66), and active variants thereof.

In a still further embodiment, the Frizzled domain component comprises apro-Frz polypeptide selected from the group consisting of hFrz1 (SEQ IDNO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID NO: 37), hFrz4 (SEQ ID NO:38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ ID NO: 40), hFrz7 (SEQ ID NO:41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ ID NO: 43), and hFrz10 (SEQ IDNO: 44), and active variants thereof, or a pro-sFrp polypeptide selectedfrom the group consisting of sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO:46), sFRP3 (SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO:49), and active variants thereof.

In one embodiment, the Wnt antagonist comprises a Fc component derivedfrom an immunoglobulin selected from the group consisting of IgG1, IgG2,IgG3 and IgG4. In another embodiment, the Fc is derived from an IgG1immunoglobulin. In yet another embodiment the Fc sequence shown in SEQID NO: 67 or SEQ ID NO: 68.

In one embodiment, Wnt antagonist further comprises a linker connectingthe Frizzled domain component to the Fc domain. In one embodiment, thelinker is a peptide linker such as ESGGGGVT (SEQ ID NO: 69), LESGGGGVT(SEQ ID NO: 70), GRAQVT (SEQ ID NO: 71), WRAQVT (SEQ ID NO: 72), andARGRAQVT (SEQ ID NO: 73).

In particular embodiments, the Wnt antagonist comprises a polypeptideselected from the group consisting of Frz8-Fc (SEQ ID NO: 74), Frz5-Fc(SEQ ID NO: 75), Frz1-Fc (SEQ ID NO: 76), Frz2-Fc (SEQ ID NO: 77),Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ ID NO: 79), Frz6-Fc (SEQ ID NO:80), Frz7-Fc (SEQ ID NO: 81), Frz9-Fc (SEQ ID NO: 82), Frz10-Fc (SEQ IDNO: 83), sFRP1-Fc (SEQ ID NO: 84), sFRP2 (SEQ ID NO: 85), sFRP3-Fc (SEQID NO: 86), sFRP4-Fc (SEQ ID NO: 87), and sFRP5-Fc (SEQ ID NO: 88).

In one embodiment, the cell proliferative disorder is cancer such ascolon cancer, colorectal cancer, breast cancer, leukemia, gliomas, ormedulloblastomas.

DESCRIPTION OF THE FIGURES

FIG. 1 is an abbreviated summary of the canonical Wnt signaling pathwayboth in the “off” or inactive state as well as the “on” or active state.

FIG. 2 is a schematic diagram representing a Frizzled extracellulardomain linked to the Fc region of a human immunoglobulin domain.

FIGS. 3A (3A-1 and 3A-2) and 3B (3B-1 and 3B-2) is an alignment of the17 known Frizzled protein extracellular domains. FIG. 3A shows analignment of the extracellular domains of the 10 pro-Frizzled proteins(SEQ ID NOs: 35-44) and the 5 pro-sFRP proteins (SEQ ID NOs: 45-49),while FIG. 3B shows an alignment of the extracellular domains of 10mature Frizzled proteins (SEQ ID NOs: 50-59), and 5 mature sFRP proteins(SEQ ID NOs: 60-64), as well as the extracellular domains of the matureRor proteins (SEQ ID NOs: 65-66). Similar residues are boxed in gray,identical residues are indicated by asterisks. Similar residues aregrouped as acidic, basic, polar and non-polar. In FIG. 3B, the minimalCRD (ECD) domains are indicated between the two boxed arrowed lines (SEQID NOs: 18-34).

FIG. 4A-4B shows the sequences of the Frz (156)-Fc and Frz (173)-Fcchimeric constructs. FIG. 4A shows the longer Frz (173)-Fc sequence (SEQID NO: 113). Shown in bold (i.e., first 24 N-terminal amino acidresidues) is the leader signal sequence. Residues 25-27 are alanineresidues that may be present or absent in the mature protein. Shown inboxed text (i.e., residues 157-173) are the additional sequences of theFrz8 receptors that distinguish the longer (Frz173) from the shorter(Frz156) chimeric constructs. The linker sequence (i.e., residues174-182) is underlined, while the Fc domain sequence is shown in italics(i.e., residues 183-409). FIG. 4B shows the shorter Frz (156)-Fc (SEQ IDNO: 74). In bold (i.e., first 24 N-terminal amino acid residues) is theleader signal sequence. Residues 25-27 are alanine residues that may bepresent or absent in the mature protein. The linker sequence (i.e.,residues 157-164) is underlined, while the Fc domain sequence is shownin italics (i.e., residues 165-391).

FIG. 5A-5H shows the nucleic acid sequence encoding several Wntantagonist chimeric constructs (Frz1-Fc (SEQ ID NO: 115), Frz2-Fc (SEQID NO: 116), Frz3-Fc (SEQ ID NO: 117), Frz4-Fc (SEQ ID NO: 118), Frz5-Fc(SEQ ID NO: 119), Frz6-Fc (SEQ ID NO: 120), Frz7-Fc (SEQ ID NO: 121),Frz8-Fc (SEQ ID NO: 122), Frz9-Fc (SEQ ID NO: 123), Frz10-Fc (SEQ ID NO:124), sFRP1-Fc (SEQ ID NO: 125), sFRP2-Fc (SEQ ID NO: 126), sFRP3-Fc(SEQ ID NO: 127), sFRP4-Fc (SEQ ID NO: 128), and sFRP5-Fc (SEQ IDNO:129)).

FIG. 6A-6E shows the full length amino acid sequences of the human Frz,sFRP, and Ror proteins.

FIG. 7A-7C shows the amino acid sequences of several Wnt antagonistchimeric constructs (Frz1-Fc (SEQ ID NO: 76), Frz2-Fc (SEQ ID NO: 77),Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ ID NO: 79), Frz5-Fc (SEQ ID NO:75), Frz6-Fc (SEQ ID NO: 80), Frz7-Fc (SEQ ID NO: 81), Frz8-Fc (SEQ IDNO: 74), Frz9-Fc (SEQ ID NO: 82), Frz10-Fc (SEQ ID NO: 83), sFRP1-Fc(SEQ ID NO: 84), sFRP2-Fc (SEQ ID NO: 85), sFRP3-Fc (SEQ ID NO: 86),sFRP4-Fc (SEQ ID NO: 87), and sFRP5-Fc (SEQ ID NO: 88)). The bold textfor Frz1-Fc (first 28 N-terminal amino acid residues), Frz2-Fc (first 31N-terminal amino acid residues), Frz3-Fc (first 31 N-terminal amino acidresidues), Frz4-Fc (first 31 N-terminal amino acid residues) Frz5-Fc(first 31 N-terminal amino acid residues), and sFRP3-Fc (first 31N-terminal amino acid residues) indicates a non-native leader sequence.The linker is underlined and the Fc domain, following the linker, isshown in italics.

FIG. 8 shows an alignment of Frizzled extracellular domains where blackshows conserved residues across all receptors and gray representsresidues conserved across homologous groups.

FIG. 9 shows Frizzleds grouped into families based on both full-lengthand extracellular domain sequence identities.

FIG. 10 depicts samples of purified Frizzled-Fc fusion proteinsexpressed and purified from CHO cells. Samples were separated onnon-reducing SDS-PAGE gels and imaged by Coomassie staining.

FIG. 11A-11B show a comparison of serum stability of the two differentFrz8-Fc chimeras Frz8(173)-Fc and Frz(156)-Fc. FIG. 11A is an immunoblotfor human FC used to detect the chimeric proteins present at increasingtime points in serum of athymic nude mice injected with the chimeras.

FIG. 11B shows the Wnt inhibitory activity of the chimeric proteinsassayed by measuring TOPglow activity shown on the Y axis as relativeluciferase activity.

FIG. 12 is a graph of tumor volume over time resulting from treatmentwith Frz8(173)-Fc chimera.

FIG. 13A-13D shows pharmacokinetic (PK) data for Frz8-Fc followingadministration of a single dose of the protein. FIG. 13A is animmunoblot of a neat serum from mice treated with Frz8-Fc showingdetection in serum at 7 days and beyond from both 20 or 5 mg/kg I.V. or20 mg/kg I.P.

FIGS. 13B and 13C are a graphical summary of Frz8-Fc serum levels asdetermined from the pharmacokinetic study. FIG. 13D is a summary of theparameters for a biphasic model of Frz8-Fc pharmacokinetics.

FIG. 14A-14B demonstrate the enhanced ability of Frz8-ECD to block Wnt3asignaling when linked to a dimeric Fc domain. FIG. 14A is an IC₅₀ graphof a Wnt3a inhibition assay of two different preparations ofFrz8(156)-FC. FIG. 14B is a gel confirming the purity of the isolatedFrz8(156) CRD (ECD). Shown are: (a) non-reduced Frz8 ECD (Lane 1); (b)molecular weight markers (Lane 2); and reduced Frz8 ECD (Lane 3).

FIG. 15A-15B demonstrates direct binding of Wnt3a to the Frz8-Fcchimera. FIG. 15A is BIAcore sensogram demonstrating binding of purifiedsoluble Wnt3a to immobilized Frz8-Fc. FIG. 15B is an immunoprecipitationof a purified soluble Wnt3a by immobilized Frz8-Fc.

FIG. 16A-16C demonstrates direct binding of several Frz-Fc chimeras toWnt ligands as measured using the OCTET™ system. FIG. 16A shows datafrom the binding of Wnt3a to the Frz1-Frz10-Fc chimeras, FIG. 16B showsdata from the binding of Wnt3a to sFRP-Fc chimeras. FIG. 16C shows datafrom the binding of Wnt5a to the Frz1-Frz10-Fc chimeras and sFRP-Fcchimeras.

FIG. 17A-17B shows the effect of the Wnt antagonists on Wnt-stimulatedcells transiently transfected with TOPglow luciferase TCF reporterplasmid. FIG. 17A shows cells stimulated with Wnt3a and FIG. 17B showscells stimulated with Wnt-5a. Cells to be treated with Wnt5a weretransfected with Frz4 and Lrp5 in addition to the reporter. 293 (humankidney) cells were activated with 100 ng/ml Wnt3a or 1 ug/ml Wnt5a.Cells then left untreated, treated with control Fc, or treated withpurified Frz-Fc protein in PBS and assayed for luciferase response.

FIG. 18 shows inhibition of Wnt signaling by the Wnt antagonists in U2OS(human osteosarcoma) cells stably transfected with a luciferase TCFreporter plasmid. Initial Wnt signaling in cells was obtained with Wnt3aactivation.

FIG. 19A-19B shows the effect of Frz8-Fc on expression of Wnt-targetgenes in cultured teratoma cells and tumor xenografts. FIG. 19A showsexpression of Wnt-target genes in PA-1 cell lines treated with Wnt3a andFrz8-Fc. RNA isolated from PA-1 cells that were treated with Wnt3a,Frz8-Fc, or control Fc protein was subject to microarray analysis andthe change in expression levels of the indicated genes in response toexogenously added Wnt3a, Frz8-Fc, and control Fc protein was plotted.Columns, mean expression level from three wells; bars, standard error(S). FIG. 19B shows the relative expression of Wnt target genes APCDD1,Gad-1, and Fzd5 in NTera-2 tumors from mice given PBS, CD4-Fc, orFrz8-Fc relative to PBS control. The data represents the mean expressionlevel from the indicated number of tumors and is representative of atleast two independent qRT-PCR experiments done in duplicate. Regulationof expression of each gene by the addition of purified Wnt3a to thecorresponding cultured cells is also presented.

FIG. 20 shows the accession number and sequence of primers and probesused for real-time quantitative PCR analysis of gene expression shown inFIG. 19 (Example 9).

FIG. 21 is a linear schematic describing the vector construct used inthe transfection to create the Wnt animal model.

FIG. 22A-22B illustrates the efficacy of Frz8-Fc against MMTV-Wnt tumortransplants in athymic nude mice by intraperitoneal (IP) dosing. FIG.22A is a graph showing data from nude mice hosting MMTV-Wnt-1 tumortransplants were administered PBS, CD4-Fc (10 mg/kg/day) or Frz8-Fc (10mg/kg/day) by intraperitoneal injection twice weekly. Mean tumor volumeis plotted over time and the treatment days are indicated by arrows onthe X-axis. FIG. 22B is tabular summary of mean tumor volume and mean %change in tumor volume over time in the four treatment groups.

FIG. 23A-23B illustrates the efficacy of Frz8-Fc against MMTV-Wnt tumortransplant in athymic nude mice by intravenous (IV) dosing. FIG. 23A isa graph showing data from nude mice hosting MMTV-Wnt-1 tumor transplantswere administered PBS, CD4-Fc (10 mg/kg/day) or Frz8-Fc (10 mg/kg/day)by intravenous injection three times weekly. Mean tumor volume isplotted over time and the treatment days are indicated by arrows on theX-axis. FIG. 23B is a tabular summary of mean tumor volume and mean %change in tumor volume over time in the four treatment groups.

FIG. 24A-24B is a bar graph showing the Wnt signaling antagonistactivity in the TOPglow assay of various Wnt antagonists in serumisolated from the MMTV Wnt tumor study. The X-axis samples appear ingroups A-E (FIG. 24A) or A-F (FIG. 24B) according to treatment, mousestudy number and dilution. The relative luciferase activity in theTOPGLOW gene reporter assay is shown on the Y-axis. All samples aretreated with ˜40 ng/ml purified Wnt3a except for NA (control). All otherprotein controls are present in the medium at 5 μg/ml. FIG. 24A showsthe testing results of serum isolated from IP treated mice, while the IVtreated ones appear in FIG. 24B.

FIG. 25A-25B shows Wnt signaling antagonist activity in the TOPglowassay of various Wnt antagonists in the indicated teratacarcinoma celllines in the absence (FIG. 25A) or presence (FIG. 25B) of exogenouslyadded Wnt3a. For each cell line, activity was expressed relative to thatobserved in the absence of any treatment (NA); representative of atleast two independent experiments. Relative luciferase activity (Y-axis)were measured from TOPglow assays from various cancer cell lines in thepresence or absence or Wnt inhibitors.

FIG. 26A-26E demonstrates the anti-tumor efficacy of Frz8 (156)-Fctreatment on the growth of NTera2 tumor xenografts in athymic nude mice.FIG. 26A is procedural flow chart, while FIG. 26B is a graph plottingmean tumor volume over time, wherein the treatment days are indicated byarrows on the X-axis. FIG. 26C is a bar graph plotting the mean tumorweights at sacrifice of all animals in the group at day 20 of the study.FIGS. 26D and 26E are tabular summaries of mean tumor volume and mean %change in tumor volume, respectively.

FIG. 27 is a bar graph showing Wnt signaling antagonist activity ofserum isolated from various animals in the NTera2 tumor study asdetermined by the TOPglow assay. The Y-axis shows relative luciferaseactivity (Y-axis) from the TOPglow assay for the controls and Frz8-FcWnt antagonist. No additional purified Wnt or Wnt conditioned media wasadded to the cells.

FIG. 28A-28E shows the anti-tumor efficacy of Frz8 (156)-Fc treatment onthe growth of PA-1 tumor xenografts in athymic nude mice. FIG. 28A is aprocedural flow chart, while FIG. 28B is a graph plotting mean tumorvolume over time. FIG. 28C is a graph of mean tumor weight at sacrifice.The mean tumor weight±SEM is plotted as a function of the group. FIGS.28D and 28E are tabular summaries of mean tumor volume and mean % changein tumor volume, respectively.

FIG. 29 shows Wnt signaling inhibition in mice treated with Frz8-Fc orFrz5-Fz as determined by the TOPglow assay. The Y-axis shows relativeluciferase activity (Y-axis) from the TOPglow assay for the controls andFrz8-Fc and Frz5-Fc Wnt antagonists.

FIG. 30A-30B shows the reduced Axin2 expression in Frz8-Fc and Frz5-Fztreated tumor with FIG. 30A showing expression normalized to expressionof GAPDH and FIG. 30B showing expression normalized to expression ofrp119.

FIG. 31A-1 to 31B-3 shows immunohistochemistry (IHC) photomicrographsfor IHC staining of β-catenin and demonstrate that Frz8-Fc treatment onregenerative tissues such as intestine and skin appear normal. FIG. 31Ashows IHC for β-catenin in small intestine of PBS (A-1) control protein(A-2) and Frz8-Fc (A-3) treated mice. FIG. 31B shows IHC for β-cateninin skin of PBS (B-1) control protein (B-2) and Frz8-Fc (B-3) treatedmice.

FIG. 32A-1 to 32C is an illustration of active Wnt signaling in humanbreast cancer. FIG. 32A shows Wnt-1 expression (as shown by in vitrohybridization) in normal (A-1), low grade (A-2) and high grade (A-3)human breast tumor initially reported in Wong et al., J. Pathol. 196:145 (2002). FIG. 32B shows nuclear (B-1) and cytoplasmic (B-2)localization (as shown by IHC) of β-catenin in breast cancer patients.Also shown is a Kaplan-Meier survival plot (B-3) showing patientsurvival probability that correlates with the indicated β-cateninexpression pattern. This data was initially reported in Lin et al.,P.N.A.S. (USA) 97(8): 4262-66 (2000). FIG. 32C is a microarray analysisof Wnt-1 expression in a normal breast from a patient without cancer incomparison with tissue isolated from a patient with infiltrating ductalcarcinoma, her-2 negative.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

A “Wnt protein” is a ligand of the Wnt signaling pathway component whichbinds to a Frizzled receptor so as to activate Wnt signaling Specificexamples of Wnt proteins include at least 19 members, including: Wnt-1(RefSeq.: NM_005430), Wnt-2 (RefSeq.: NM_003391), Wnt-2B (Wnt-13)(RefSeq.: NM_004185), Wnt-3 (ReSeq.: NM_030753), Wnt3a (RefSeq.:NM_033131), Wnt-4 (RefSeq.: NM_030761), Wnt-5A (RefSeq.: NM_003392),Wnt-5B (RefSeq.: NM_032642), Wnt-6 (RefSeq.: NM_006522), Wnt-7A(RefSeq.: NM_004625), Wnt-7B (RefSeq.: NM_058238), Wnt-8A (RefSeq.:NM_058244), Wnt-8B (RefSeq.: NM_003393), Wnt-9A (Wnt-14) (RefSeq.:NM_003395), Wnt-9B (Wnt-15) (RefSeq.: NM_003396), Wnt-10A (RefSeq.:NM_025216), Wnt-10B (RefSeq.: NM_003394), Wnt-11 (RefSeq.: NM_004626),Wnt-16 (RefSeq.: NM_016087)). While each member has varying degrees ofsequence identity, each contain 23-24 conserved cysteine residues whichshow highly conserved spacing. McMahon, A P et al., Trends Genet. 8:236-242 (1992); Miller J R., Genome Biol. 3(1): 3001.1-3001.15 (2002).For purposes of this invention, a Wnt protein and active variantsthereof is a protein that binds to a Frizzled ECD or the CRD componentof such an Frz ECD.

A “Frizzled” (Frz) protein is a Wnt signaling pathway component that isa seven-pass transmembrane receptors that binds to a Wnt protein, andfurther complexes with other membrane-associated Wnt signalingcomponents, so as to transmit Wnt signaling to downstream intracellularcomponents. Frz proteins include Frz1, Frz2, Frz3, Frz4, Frz5, Frz6,Frz7, Frz8, Frz9, and Frz10. Examples of human full length Frz proteinsare hFrz1 (NP_003496) (SEQ ID NO: 1), hFrz2 (NP_001457) (SEQ ID NO: 2),hFrz3 (NP_059108) (SEQ ID NO: 3), hFrz4 (NP_036325) (SEQ ID NO: 4),hFrz5 (NP_003459) (SEQ ID NO: 5), hFrz6 (NP_003497) (SEQ ID NO: 6),hFrz7 (NP_003498) (SEQ ID NO: 7), hFrz8 (NP_114072) (SEQ ID NO: 8),hFrz9 (NP_003499) (SEQ ID NO: 9), and hFrz10 (NP_009128) (SEQ ID NO: 10)(FIGS. 6A-6C).

A “secreted Frizzled related protein” (sFRP) is a Wnt signaling pathwaycomponent that is a secreted extracellular polypeptide that binds to aWnt protein. sFRP proteins include sFRP1, sFRP2, sFRP3, sFRP4, andsFRP5. Examples of human full length sFRP proteins are sFRP1 (NP_003003)(SEQ ID NO: 11), sFRP2 (NP_003004) (SEQ ID NO: 12), sFRP3 (NP_001454)(SEQ ID NO: 13), sFRP4 (NP_003005) (SEQ ID NO: 14), and sFRP5(NP_003006) (SEQ ID NO: 15) (FIGS. 6C-6D).

The “Ror” protein, includes the mammalian homologs, Ror1 and Ror2, whichare characterized by extracellular Frizzled-like cysteine-rich domains(CRDs) as well as membrane proximal kringle domains. Ror proteins playcrucial roles in developmental morphogenesis and are associated withdifferent components of the cytoskeleton. Ror1 co-localizes with F-actinalong stress fibers, while Ror2 partially colocalizes with microtubules.Ror1 and Ror2 share about 58% overall sequence identity. Ror2 associateswith the melanoma-associated antigen (MAGE) family protein Dlxin-1 andregulates its intracellular distribution. Ror1 proteins include Ror1 andRor2. Examples of human full length Ror proteins are hRor1 (NP_005003)(SEQ ID NO: 16), and hRor2 (NP_004551) (SEQ ID NO: 17) (FIGS. 6D-6E).

A “Frz domain component” is a polypeptide derived from a Frz protein, asFRP protein, a Ror protein, or other protein, that is capable ofbinding with a Wnt protein. A polypeptide “derived from” a protein meansa polypeptide that has an amino acid sequence that can be found withinthe reference protein sequence or within the sequence of active variantsof the protein. Examples of a Frz domain component include a minimalcysteine rich domain (CRD) of an extracellular domain “CRD (ECD)” of aFrz protein, a sFRP protein, or a Ror protein, such as the CRD (ECD) ofFrz1, Frz2, Frz3, Frz4, Frz5, Frz6, Frz7, Frz8, Frz9, Frz10, sFRP1,sFRP2, sFRP3, sFRP4, sFRP5, Ror1, or Ror2, and active variants thereof.The CRD (ECD) is a conserved structural motif of 100 to 250 amino acidsand is defined by 10 highly conserved cysteines. Particular examples ofhuman CRD (ECD)s are shown in boxed text in FIG. 3B and presented as SEQID NOs: hFrz1 (SEQ ID NO: 18), hFrz2 (SEQ ID NO: 19), hFrz3 (SEQ ID NO:20), hFrz4 (SEQ ID NO: 21), hFrz5 (SEQ ID NO: 22), hFrz6 (SEQ ID NO:23), hFrz7 (SEQ ID NO: 24), hFrz8 (SEQ ID NO: 25), hFrz9 (SEQ ID NO:26), hFrz10 (SEQ ID NO: 27), sFRP1 (SEQ ID NO: 28), sFRP2 (SEQ ID NO:29), sFRP3 (SEQ ID NO: 30), sFRP4 (SEQ ID NO: 31), sFRP5 (SEQ ID NO:32), hRor1 (SEQ ID NO: 33), and hRor2 (SEQ ID NO: 34).

Additional examples of a Frz domain component include a pro-Frz domainderived from a pro-Frz or pro-sFRP protein such as Frz1, Frz2, Frz3,Frz4, Frz5, Frz6, Frz7, Frz8, Frz9, Frz10, sFRP1, sFRP2, sFRP3, sFRP4,or sFRP5, and active variants thereof. Particular examples of humanpro-Frz domains are shown in FIG. 3A and presented as SEQ ID NOs: hFrz1(SEQ ID NO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID NO: 37), hFrz4(SEQ ID NO: 38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ ID NO: 40), hFrz7(SEQ ID NO: 41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ ID NO: 43), hFrz10(SEQ ID NO: 44), sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO: 46), sFRP3(SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO: 49).

Additional examples of a Frz domain component include a mature Frzdomain derived from a mature Frz, sFRP, or Ror protein, such as Frz1,Frz2, Frz3, Frz4, Frz5, Frz6, Frz7, Frz8, Frz9, Frz10, sFRP1, sFRP2,sFRP3, sFRP4, sFRP5, Ror1, or Ror2 and active variants thereof.Particular examples of human mature Frz domains are shown in FIG. 3B andpresented as SEQ ID NOs: hFrz1 (SEQ ID NO: 50), hFrz2 (SEQ ID NO: 51),hFrz3 (SEQ ID NO: 52), hFrz4 (SEQ ID NO: 53), hFrz5 (SEQ ID NO: 54),hFrz6 (SEQ ID NO: 55), hFrz7 (SEQ ID NO: 56), hFrz8 (SEQ ID NO: 57),hFrz9 (SEQ ID NO: 58), hFrz10 (SEQ ID NO: 59), sFRP1 (SEQ ID NO: 60),sFRP2 (SEQ ID NO: 61), sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63),sFRP5 (SEQ ID NO: 64), hRor1 (SEQ ID NO: 65), and hRor2 (SEQ ID NO: 66).

A “Wnt antagonist” is a chimeric polypeptide comprising a Frz domaincomponent and an immunoglobulin Fc domain that binds to a Wnt proteinand is active by attenuating cellular Wnt signaling, or a physiologicalsymptom resulting therefrom.

In certain embodiments, the Fc domain is a human IgG1, IgG2, IgG3 orIgG4 Fc domain. In one embodiment, the Fc domain is a human IgG1 Fcdomain. Specific examples of Fc domains are shown in FIGS. 4, 5, andFIG. 7 and in SEQ ID NO: 67 and SEQ ID NO: 68.

In some embodiments, the Frz domain component and the Fc domain arefused by a linker. The term “linker” refers to a component that tetherstogether the Frz domain component to the Fc domain. Linkers that aresuitable for use in the invention exhibit minimal or no interferencewith expression, secretion and folding of the protein domains of the Wntantagonist molecules and provide minimal or no interference with eitherthe effector function of the Fc domain or Wnt protein interactionfunction of the Frz domain (e.g., binding to a Wnt protein) throughsteric or other means. In particular embodiments, the linker is shortpeptide sequence. A linker sequence may also include additional aminoacid residues from either the Frz domain component or Fc domain outsidethe minimal residues needed for activity. Preferred linkers will alsoprovide for good serum stability and are resistant to protease cleavage.Specific examples of useful linkers appear in FIG. 4, FIG. 5, and FIG.7, including the sequences ESGGGGVT (SEQ ID NO: 69), LESGGGGVT (SEQ IDNO: 70), GRAQVT (SEQ ID NO: 71), WRAQVT (SEQ ID NO: 72), and ARGRAQVT(SEQ ID NO: 73). As noted above, these linkers may include additionalamino acid residues from either the Frz domain component or the Fcdomain outside the minimal residues needed for activity. These linkersmay also comprise additional amino acid residues other than those fromthe Frz domain component or Fc domain component.

A “Wnt signaling pathway component” is a component that transduces asignal originating from an interaction between a Wnt protein and an Frzreceptor. As the Wnt signaling pathway is complex, and involvesextensive feedback regulation, there are numerous and likely not yetdiscovered members of the Wnt signaling pathway. Example Wnt signalingpathway components include the membrane associated proteins LRP5 andLRP6, Axin, and Dishevelled, the extracellular Wnt interactive proteinssFRP, WIF-1, the LRP inactivating proteins Dkk and Krn, the cytoplasmicprotein β-catenin, members of the β-catenin “degradation complex” APC,GSK3β, CKIα and PP2A, the nuclear transport proteins APC, pygopus andbcl9/legless, and the transcription factors TCF/LEF, Groucho and varioushistone acetylases such as CBP/p300 and Brg-1.

A “Wnt-mediated disorder” is a disorder, condition, or disease statecharacterized by aberrant Wnt signaling. In a specific aspect, theaberrant Wnt signaling is a level of Wnt signaling in a cell or tissuesuspected of being diseased that exceeds the level of Wnt signaling in asimilar non-diseased cell or tissue. In a specific aspect, aWnt-mediated disorder includes cancer.

The term “cancer” refers to the physiological condition in mammals thatis typically characterized by unregulated cell growth/proliferation.Examples of cancer include, but are not limited to: carcinoma, lymphoma,blastoma, and leukemia. More particular examples of cancers include, butare not limited to: chronic lymphocytic leukemia (CLL), lung, includingnon small cell (NSCLC), breast, ovarian, cervical, endometrial,prostate, colorectal, intestinal carcinoid, bladder, gastric,pancreatic, hepatic (hepatocellular), hepatoblastoma, esophageal,pulmonary adenocarcinoma, mesothelioma, synovial sarcoma, osteosarcoma,head and neck squamous cell carcinoma, juvenile nasopharyngealangiofibromas, liposarcoma, thyroid, melanoma, basal cell carcinoma(BCC), medulloblastoma and desmoid.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

An “active” polypeptide, variant polypeptide, or fragments thereof,retain a biological activity of native or naturally-occurring componentof the active polypeptide. Biological activity refers to a functionmediated by the native or naturally-occurring counterpart of the activepolypeptide. For example, binding or a protein-protein interactionconstitutes a biological activity. In a specific sense, an active Wntsignaling pathway component is one which can effectively transduce asignal through interaction with other Wnt signaling pathway components.In another specific sense, an active Wnt antagonist is one whichdetectably attenuates Wnt signaling or a physiological conditionresulting therefrom, relative to the level prior to administration ofthe Wnt antagonist.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“High stringency conditions”, as defined herein, may be identified bythose that: (1) employ low ionic strength and high temperature forwashing, for example 0.015 M sodium chloride/0.0015 M sodiumcitrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) overnighthybridization in a solution that employs 50% formamide, 5×SSC (0.75 MNaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA(50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with a 10minute wash at 42° C. in 0.2×SSC (sodium chloride/sodium citrate)followed by a 10 minute high-stringency wash consisting of 0.1×SSCcontaining EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” refers to a polypeptide that is fused to a“tag polypeptide.” The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the polypeptide to which itis fused. The tag polypeptide preferably also is fairly unique so thatthe antibody does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least six amino acidresidues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues). Exampleepitope tag sequences include HA, GD, c-myc, poly-His and FLAG.

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic disease orcondition or disorder. Those in need of treatment include those alreadywith the disorder as well as those prone to having the disorder or thosein whom the disorder is to be prevented (prophylaxis). When theWnt-mediated disorder is cancer, a subject or mammal is successfully“treated” or shows a reduced tumor burden if, after receiving atherapeutic amount of a Wnt antagonist according to the methods of thepresent invention, the patient shows observable and/or measurablereduction in or absence of one or more of the following: reduction inthe number of cancer cells or absence of the cancer cells; reduction inthe tumor size; inhibition (i.e., slow to some extent and preferablystop) of cancer cell infiltration into peripheral organs including thespread of cancer into soft tissue and bone; inhibition (i.e., slow tosome extent and preferably stop) of tumor metastasis; inhibition, tosome extent, of tumor growth; and/or relief to some extent, one or moreof the symptoms associated with the specific cancer; reduced morbidityand mortality, and improvement in quality of life issues. To the extentthe Wnt antagonist may prevent growth and/or kill existing cancer cells,it may be cytostatic and/or cytotoxic. Reduction of these signs orsymptoms may also be felt by the patient.

The above parameters for assessing successful treatment and improvementin the disorder are readily measurable by routine procedures familiar toa physician. For cancer therapy, efficacy can be measured, for example,by assessing the time to disease progression (TDP) and/or determiningthe response rate (RR). Metastasis can be determined by staging testsand by bone scan and tests for calcium level and other enzymes todetermine spread to the bone. CT scans can also be done to look forspread to the pelvis and lymph nodes in the area. Chest X-rays andmeasurement of liver enzyme levels by known methods are used to look formetastasis to the lungs and liver, respectively. Other routine methodsfor monitoring the disease include transrectal ultrasonography (TRUS)and transrectal needle biopsy (TRNB).

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is cyclic, or subject toperiodic interruptions, as opposed to continuous or consecutive.

“Mammal” refers to any animal classified as a mammal, including humans,domestic and farm animals, and zoo, sports, or pet animals, such asdogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

An “effective amount” of a Wnt antagonist is an amount sufficient tocarry out a specifically stated purpose. An “effective amount” may bedetermined empirically and in a routine manner, in relation to thestated purpose.

The term “therapeutically effective amount” refers to an amount of a Wntantagonist effective to “treat” a Wnt-mediated disorder in a subject ormammal. In the case of cancer, the therapeutically effective amount ofthe drug may reduce the number of cancer cells; reduce the tumor size;inhibit (i.e., slow to some extent and preferably stop) cancer cellinfiltration into peripheral organs; inhibit (i.e., slow to some extentand preferably stop) tumor metastasis; inhibit, to some extent, tumorgrowth; and/or relieve to some extent one or more of the symptomsassociated with the cancer. See the definition herein of “treating”. Tothe extent the drug may prevent growth and/or kill existing cancercells, it may be cytostatic and/or cytotoxic.

A “growth inhibitory amount” of a Wnt antagonist is an amount capable ofinhibiting the growth of a cell, especially tumor, e.g., cancer cell,either in vitro or in vivo. A “growth inhibitory amount” of a Wntantagonist for purposes of inhibiting neoplastic cell growth may bedetermined empirically and in a routine manner.

A “cytotoxic amount” of a Wnt antagonist is an amount capable of causingthe destruction of a cell, especially tumor, e.g., cancer cell, eitherin vitro or in vivo. A “cytotoxic amount” of a Wnt antagonist forpurposes of inhibiting neoplastic cell growth may be determinedempirically and in a routine manner.

The terms “antibody” and “immunoglobulin” are used interchangeably, andin the broadest sense, including monoclonal antibodies (e.g., fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies exhibiting the desired biological activity) and may alsoinclude certain antibody fragments, as described herein in greaterdetail. An antibody can be chimeric, human, humanized or affinitymatured.

The light chain from any vertebrate species can be assigned to one oftwo clearly distinct types, called kappa (κ) and lambda (λ), based onthe amino acid sequences of their constant domains. Depending on theamino acid sequence of the constant domain of their heavy chains(C_(H)), immunoglobulins can be assigned to different classes orisotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG,and IgM, having heavy chains designated α, δ, ε, γ, and μ, respectively.The γ and α classes are further divided into subclasses on the basis ofrelatively minor differences in C_(H) sequence and function, e.g.,humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1,and IgA2. The subunit structures and three-dimensional configurations ofdifferent classes of immunoglobulins are well known and describedgenerally in, for example, Abbas et al., Cellular and Molecular Biology,4^(th) Ed. (2000). An antibody may be part of a larger fusion molecule,formed by covalent or non-covalent associated of the antibody with oneor more other proteins or peptides.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments, diabodies, linear antibodies (U.S. Pat. No. 5,641,870);Zapata et al., Protein Eng. 8(10): 1057-1062 (1995), single chainantibody molecules and multispecific antibodies formed from antibodyfragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire light chain along with the variableregion domain of the heavy chain (V_(H)), and the first constant domainof one heavy chain (C_(H)1). Each Fab fragment is monovalent withrespect to antigen binding, i.e., it has a single antigen-binding site.Pepsin treatment of an antibody yields a single large F(ab′)₂ fragmentwhich roughly corresponds to two disulfide linked Fab fragments havingdivalent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having additionalfew residues at the carboxy terminus of the C_(H)1 domain including oneor more cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both heavychains held together by disulfides. The effector functions of antibodiesare determined by sequences in the Fc region, which region is also thepart recognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogenous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. In contract to polyclonal antibodypreparations that typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen.

The term “chimeric” antibody, specifically included within thedefinition of monoclonal antibody, means antibodies in which a portionof the heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences derived from another species or belonging toanother antibody class or subclass, as well as fragment of suchantibodies, so long as they exhibit the desired biological activity U.S.Pat. No. 4,816,567; Morrison et al., P.N.A.S. USA 81: 6851-6855 (1984).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, Vaswani andHamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris,Biochem. Soc. Trans. 23: 1035-1038 (1995); Hurle and Gross, Curr. Op.Biotech. 5: 428-433 (1994).

“Polynucleotide” or “nucleic acid” are used interchangeably herein, andrefer to polymers of nucleotides of any length, including, but are notlimited to DNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a polymer by DNA or RNApolymerase, or by a synthetic reaction. A polynucleotide may comprisemodified nucleotides, such as methylated nucleotides and their analogs.If present, modification to the nucleotide structure may be importedbefore or after assembly of the polymer. The sequence of nucleotides maybe interrupted by non-nucleotide components. A polynucleotide may befurther modified after synthesis, such as by conjugation with a label.Other types of modifications include, for example, “caps”, substitutionof one or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example: uncharged linkages(e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.); charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.); pendant moieties, such as, for example,proteins (e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.); intercalators (e.g., acridine, psoralen, etc.);chelators (e.g., metals, radioactive metals, boron, oxidative metal,etc.), alkylators, modified linkages (e.g., alpha anomeric nucleicacids, etc.). Further, any of the hydroxyl groups ordinarily present inthe sugars may be replaced, for example, by phosphonate groups,phosphate groups, protected by standard protecting groups, or activatedto prepare additional linkages to additional nucleotides, or may beconjugated to solid or semi-solid supports. The 5′ and 3′ terminal OHcan be phosphorylated or substituted with amines or organic cappinggroup moieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose,pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs andabasic nucleoside analogs such as methyl riboside. One or morephosphodiester linkages may be replaced by alternative linking groups.These alternative linking groups include, but are not limited to,embodiments wherein phosphate is replace by P(O)—S-(thioate),P(S)—S-(dithioate)-, (O)NR₂-amidate, P(O)R, P(O)OR′, CO orCH₂-(formacetal), in which each R or R′ is independently H orsubstituted or unsubstituted C₁₋₂₀ alkyl, optionally containing anether, aryl, alkenyl, cycloalkenyl or aralkyl linkage. Not all linkagesin a polynucleotide need be identical. The preceding description appliesto all polynucleotides referred to herein, including RNA and DNA.

The term “peptide” generally refers to a contiguous and relatively shortsequence of amino acids linked by peptidyl bonds. Typically, but notnecessarily, a peptide has length of about 2 to 50 amino acids, 4-40amino acids or 10-30 amino acids. Although the term “protein” generallyrefers to longer forms of a “polypeptide,” the two terms can be and areused interchangeably in some contexts herein, and refer to amino acidsequences that are generally longer and perhaps more complex (e.g.,multiple sequence, secondary and higher structure).

A “region” of a polypeptide is a contiguous sequence of 2 or more aminoacid residues. In alternative embodiments, a region is at least about 3,5, 10, 15 or more contiguous amino acid residues.

“C-terminal region”, “C-terminal sequence” and variations thereof, asused herein, refer to an amino acid sequence that is located at or inclose proximity to the C-terminal (generally 3′) end. Generally, thesequence includes an amino acid that has a free carboxyl group. In oneembodiment, a C-terminal regions or sequence refers to a region of apolypeptide that includes about 1-15 residues located closest to theC-terminus.

“N-terminal region”, “N-terminal sequence”, and variations thereof, asused herein, refer to an amino acid sequence that is located at or inclose proximity to the N-terminal (generally 5′) end. Generally, thesequence includes an amino acid that has free amino group. In oneembodiment, an N-terminal region or sequence refers to a region of apolypeptide that includes about 1-15 residues located closest to the Nterminus of the polypeptide.

“Internal region” or “internal sequence”, and variations thereof, referto an amino acid sequence that is located within a polypeptide and isflanked on both its N- and C-termini by one or more amino acids that arenot part of the sequence. Generally, the sequence does not include anamino acid with either a free carboxyl or amino group.

A “ligand” refers to a naturally-occurring or synthetic molecule ormoiety that is capable of a binding interaction with a specific site ona protein or other molecule, such as a receptor. A Wnt ligand is amolecule that specifically interacts with a Frizzled receptor. A“receptor” is often, but need not be located on the cell surface ormembrane.

A “fusion protein” refers to a polypeptide having two portionscovalently linked together, where each of the portions is derived fromdifferent proteins. The two portions may be linked directly by a singlepeptide bond or through a peptide linker containing one or more aminoacid residues. Generally, the two portions and the linker will be inreading frame with each other and are produced using recombinanttechniques.

A Wnt antagonist that “inhibits the growth of tumor cells” or a “growthinhibitory” Wnt antagonist is one which results in measurable growthinhibition of tumor cells having aberrant Wnt signaling activity.Preferred growth inhibitory Wnt antagonists inhibit growth of tumorcells having aberrant Wnt signaling activity by greater than 20%,preferably from about 20% to about 50%, and even more preferably, bygreater than 50% (e.g., from about 50% to about 100%) as compared to theappropriate control, the control typically being cancer cells nottreated with the Wnt antagonist molecule being tested. In oneembodiment, growth inhibition can be measured at a Wnt antagonistconcentration of about 0.1 to 30 μg/ml or about 0.5 nM to 200 nM in cellculture, where the growth inhibition is determined 1-10 days afterexposure of the tumor cells to the Wnt antagonist. Growth inhibition oftumor cells in vivo can be determined in various ways such as isdescribed in the Experimental Examples section below. The Wnt antagonistis growth inhibitory in vivo if administration of the Wnt antagonist atabout 1 μg/kg to about 100 mg/kg body weight results in reduction intumor size or cell proliferation within about 5 days to 3 months fromthe first administration of the antibody, preferably within about 5 to30 days. In a specific aspect, the tumor size is reduced relative to itssize at the start of therapy.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

A Wnt antagonist molecule which “induces cell death” is one which causesa viable cell to become nonviable. The cell is one having aberrant Wntsignaling activity as compared to a normal cell of the same tissue type.Preferably, the cell is a cancer cell, as defined herein. Cell death invitro may be determined in the absence of complement and immune effectorcells to distinguish cell death induced by antibody-dependentcell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity(CDC). Thus, the assay for cell death may be performed using heatinactivated serum (i.e., in the absence of complement) and in theabsence of immune effector cells. To determine whether the Wntantagonist is able to induce cell death, loss of membrane integrity asevaluated by uptake of propidium iodide (PI), trypan blue (see Moore etal. Cytotechnology 17:1-11 (1995)) or 7AAD can be assessed relative tountreated cells. Preferred cell death-inducing antibodies, oligopeptidesor other organic molecules are those which induce PI uptake in the PIuptake assay in BT474 cells.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibody,oligopeptide or other organic molecule so as to generate a “labeled”antibody, oligopeptide or other organic molecule. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, enzymes and fragments thereofsuch as nucleolytic enzymes, antibiotics, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof, andthe various antitumor or anticancer agents disclosed below. Othercytotoxic agents are described below. A tumoricidal agent causesdestruction of tumor cells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e. g., calicheamicin,especially calicheamicin gammall and calicheamicin omegaI1 (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, tnoxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially a cancer cellhaving Wnt signaling activity, either in vitro or in vivo. Thus, thegrowth inhibitory agent may be one which significantly reduces thepercentage of such cells in S phase. Examples of growth inhibitoryagents include agents that block cell cycle progression (at a placeother than S phase), such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (W B Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Doxorubicin” is an anthracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

II. Description of Specific Embodiments

The Wnt antagonists described herein are capable of binding to Wntligands in vitro and are capable of inhibiting or suppressing Wntstimulated cell signaling. Additionally, the Wnt antagonists have a longin vivo half life and exhibit anti-tumor activity in vivo, inhibitingthe growth of Wnt-1 driven tumors in a mouse MMTV breast tumor model.The Wnt antagonists are also capable of inhibiting the growth in mice oftumor xenografts derived from human teratoma cell lines. Regenerativetissues taken from mice that were treated with a Wnt antagonist appearto be within physiological norms. The Wnt antagonists are also capableof inhibiting autocrine Wnt signaling in human tumor cell lines invitro.

The Frizzled receptor proteins can be grouped into families based onboth full-length and extracellular domain sequence identities. Thisgrouping is illustrated in the alignments shown in FIGS. 8 and 9. Theunderlined residues in this figure are conserved across all the Frzreceptors and the shadowed residues are conserved across homologousgroupings. The Frz proteins can be grouped into the followingfamilies 1) Frz1, Frz2, and Frz7 having a shared homology of 68-77% forthe full length sequence and 90% for the ECD; 2) Frz5 and Frz8 having ashared homology of 57% for the full length sequence and 80% for the ECD;3) Frz9 and Frz10 having a shared homology of 61% for the full lengthsequence and 74% for the ECD; 4) Frz3 and Frz6 having a shared homologyof 49% for the full length sequence and 50% for the ECD; and 5) Frz4(which exhibits a shared homology of 46% for the full length sequenceand 48% for the ECD with Frz10). The family of Frz1, Frz2, and Frz7 alsohas significant homology to Drosophila Frz1 and the family of Frz5 andFrz8 has significant homology to Drosophila Frz2, shown to beresponsible for planar cell polarity and Wnt signaling, respectively.

Wnt ligand-Frizzled binding behavior appears to cluster within Frizzledfamilies. Both Wnt3a and Wnt5a bind Frz5, Frz8, and Frz4 fastestrelative to the other Frz proteins while Wnt3a binds Frz1, Frz2, andFrz7 at a slower rate. The amplitude and linear nature of Wnt5a bindingbehavior is indicative of lower binding affinity, relative to Wnt3abinding, as determined by the OCTET™ binding assay. The presence of bothhigh affinity and low affinity receptors may confer ability for acuteand long term signaling.

The ability of the Wnt antagonists to inhibit Wnt ligand inducedsignaling also appears to cluster within Frizzled families. Both Frz5and Frz8 show complete inhibition of the Wnt3a signal and significantinhibition of the Wnt5a signal in a cell-based assay (Example 7). Frz4,Frz2, and Frz7 show significant inhibition of the Wnt3a signal. Thisfinding mirrors the observation in Drosophila that dFrz2 (with homologyto Frz5 and Frz8) strongly activates and dFrz1 (with homology to Frz1,Frz2, and Frz7) can weakly activate the Wnt pathway.

While not being bound to a particular theory of action, the datapresented herein indicate that cell-based Wnt signaling inhibition datagenerated using the Wnt antagonists correlates with data obtained bymeasuring the direct binding of Wnt ligands to the Wnt antagonists,indicating that the Wnt antagonists bind directly to Wnt ligands thusblocking them from binding the full-length Frizzled receptors on thecell. The data presented herein further provides validation that invitro activity can be used to predict in vivo Wnt signaling blockingactivity of the Wnt antagonists.

As indicated in the studies with Fz8-Fc set forth in the Examples, theWnt antagonists comprising both a Frizzled domain and an immunoglobulinFC domain surprisingly exhibit increased binding affinity to Wnt ligandover the Frizzled domain alone. For example, FIG. 14 shows that bindingaffinity increased over two orders of magnitude when the Fz ECD domainwas converted to the Fz (156)-Fc construct. The finding of the Fz(156)-Fc construct as a stable and highly efficacious Wnt signalinginhibitor, in which conjugation to Fc resulted in a two order ofmagnitude increase in binding affinity, was greatly unexpected andnon-obvious.

A. Compositions and Methods of the Invention 1. Polypeptides

The present invention is directed toward compositions and methods forthe treatment of Wnt-mediated disorders, including cancer, and forinhibiting cellular Wnt signaling. One aspect of the invention providesWnt antagonists that are chimeric molecules comprising a Frizzled (Frz)domain component and an immunoglobulin Fc domain. In particularembodiments of this aspect, the Frz domain component and Fc domain arefused through a linker. Another aspect provides for use of these Wntantagonists for inhibiting cellular Wnt signaling and for treatment ofWnt-mediated disorders, such as cancer.

In one aspect, the invention provides for Wnt antagonists that arechimeric molecules with a Frz domain component comprising a minimalcysteine rich domain (CRD) of an extracellular domain “CRD (ECD)”. TheCRD (ECD) is a conserved structural motif of 100 to 250 amino acids andis defined by 10 highly conserved cysteines. This protein domain appearsin two classes of the Wnt signaling family—the integral membrane Wntreceptor proteins known as Frizzled, and the secreted extracellularproteins known as the Frizzled related protein (sFrp).

In one aspect, the invention provides for Wnt antagonists that arechimeric molecules having a Frz domain component comprising a CRD (ECD)of a Frizzled protein such as Frz1, Frz2, Frz3, Frz4, Frz5, Frz6, Frz7,Frz8, Frz9, or Frz10. Examples of such CRD (ECD)s are provided in FIG.3B. In specific embodiments, the Frz domain component is selected fromthe group consisting of CRD (ECD)s of hFrz1 (SEQ ID NO: 18), hFrz2 (SEQID NO: 19), hFrz3 (SEQ ID NO: 20), hFrz4 (SEQ ID NO: 21), hFrz5 (SEQ IDNO: 22), hFrz6 (SEQ ID NO: 23), hFrz7 (SEQ ID NO: 24), hFrz8 (SEQ ID NO:25), hFrz9 (SEQ ID NO: 26), and hFrz10 (SEQ ID NO: 27), and activevariants thereof.

Alternatively, the Frz domain component comprises, for example, a CRD(ECD) from a secreted Frizzled related protein (sFRP) such as sFRP1,sFRP2, sFRP3, sFRP4, or sFRP5. Examples of such CRD (ECD)s are providedin FIG. 3B. In specific embodiments, the Frz domain component isselected from the group consisting CRD (ECD)s of sFRP1 (SEQ ID NO: 28),sFRP2 (SEQ ID NO: 29), sFRP3 (SEQ ID NO: 30), sFRP4 (SEQ ID NO: 31),sFRP5 (SEQ ID NO: 32), and active variants thereof.

Alternatively, the Frz domain component comprises, for example, aCRD(ECD) of the receptor tyrosine kinases Ror1 and Ror2. Examples ofsuch CRD (ECD)s are provided in FIG. 3B. In specific embodiments, theFrz domain component is selected from the group consisting of CRD (ECD)sof hRor1 (SEQ ID NO: 33), and hRor2 (SEQ ID NO: 34), and active variantsthereof.

In another aspect, the Frz domain component is a pro-Frz or pro-sFrpsequence, examples of which are shown in FIG. 3A. In specificembodiments, the Frz domain component is selected from the groupconsisting of hFrz1 (SEQ ID NO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQID NO: 37), hFrz4 (SEQ ID NO: 38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ IDNO: 40), hFrz7 (SEQ ID NO: 41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ ID NO:43), hFrz10 (SEQ ID NO: 44), sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO:46), sFRP3 (SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO:49), and active variants thereof.

In yet another aspect, the Frz domain component is derived from a matureFrz, sFRP or hRor sequence, examples of which are shown in FIG. 3B. Inspecific embodiments, the Frz domain component is selected from thegroup consisting of hFrz1 (SEQ ID NO: 50), hFrz2 (SEQ ID NO: 51), hFrz3(SEQ ID NO: 52), hFrz4 (SEQ ID NO: 53), hFrz5 (SEQ ID NO: 54), hFrz6(SEQ ID NO: 55), hFrz7 (SEQ ID NO: 56), hFrz8 (SEQ ID NO: 57), hFrz9(SEQ ID NO: 58), hFrz10 (SEQ ID NO: 59), sFRP1 (SEQ ID NO: 60), sFRP2(SEQ ID NO: 61), sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), sFRP5(SEQ ID NO: 64), hRor1 (SEQ ID NO: 65), and hRor2 (SEQ ID NO: 66), andactive variants thereof.

In particular embodiments, the Frz domain component and theimmunoglobulin Fc domain of the chimeric Wnt antagonist molecules arefused through a linker. In one embodiment, the linker is a peptidelinker. In another embodiment, the linker is selected from the groupconsisting of ESGGGGVT (SEQ ID NO: 69), LESGGGGVT (SEQ ID NO: 70),GRAQVT (SEQ ID NO: 71), WRAQVT (SEQ ID NO: 72), and ARGRAQVT (SEQ ID NO:73). Optionally, the linkers may include additional amino acid residuesfrom either the Frz domain component or the Fc domain outside theminimal residues needed for activity. These linkers may also compriseadditional amino acid residues other than those from the Frz domaincomponent or Fc domain component.

In one embodiment, the Wnt antagonist is Frz8-Fc chimera comprising aFrz8 CRD (ECD) and a Fc domain. In some embodiments, the Frz8-Fc chimerafurther comprises a linker, such as a peptide linker. In a furtherembodiment, the Frz8-Fc further comprises a leader sequence. In aparticular embodiment, the Frz domain component comprises amino acids1-156 of the Frz8 protein (SEQ ID NO: 8). In another embodiment, the Fccomponent is a human Fc. In a further embodiment, the Fc component is ahuman IgG Fc. In yet a further embodiment, the Frz8-Fc has a Frz domaincomponent comprising amino acids 1-156 of the Frz8 protein fused with alinker to a human IgG Fc. In a further embodiment, the Frz8-Fc is achimera with the amino acid sequence as shown in FIG. 4B (SEQ ID NO:74). As used in the Examples and accompanying Figures, unless otherwisenoted, “Frz8-Fc” refers to the chimera shown in FIG. 4B (SEQ ID NO: 74).

In a further embodiment, the Wnt antagonist is Frz5-Fc chimeracomprising a Frz5 CRD (ECD) and a Fc domain. In some embodiments, theFrz5-Fc chimera further comprises a linker, such as a peptide linker. Ina further embodiment, the Frz5-Fc further comprises a leader sequence.In a particular embodiment, the Frz domain component comprises aminoacids 27-155 of the Frz5 protein (SEQ ID NO: 5). In another embodiment,the Fc component is a human Fc. In a further embodiment, the Fccomponent is a human IgG Fc. In yet a further embodiment, the Frz5-Fchas a leader sequence and a Frz domain component comprising amino acids27-155 of a mature Frz5 protein fused with a linker to a human IgG Fc.In a further embodiment, the Frz5-Fc is a chimera with the amino acidsequence as shown in FIG. 7A (SEQ ID NO: 75). As used in the Examplesand accompanying Figures, unless otherwise noted, “Frz5-Fc” refers tothe chimera shown in FIG. 7A (SEQ ID NO: 75).

Similarly, further embodiments include Frz1-Fc, Frz2-Fc, Frz3-Fc,Frz4-Fc, Frz6-Fc, Frz7-Fc, Frz9-Fc, Frz-10-Fc, sFRP1-Fc, sFRP2-Fc,sFRP3-Fc, sFRP4-Fc and sFRP5-Fc chimeras comprising a Frz domaincomponent comprising a Frz CRD (ECD) from each respective Frz or sFRPprotein and a Fc component. In some embodiments, the Frz-Fc chimerafurther comprises a linker, such as a peptide linker. In furtherembodiments, these chimeras comprise a leader sequence. In someembodiments, the Fc component is a human Fc. In further embodiments, theFc component is a human IgG Fc. In yet further embodiments, thesechimeras have a leader sequence and a Frz CRD (ECD) fused with a linkerto a human IgG Fc. In further embodiment, these chimeras have the aminoacid sequences as shown in FIGS. 7A, 7B and 7C (SEQ ID NOs: 76-88). Asused in the Examples and accompanying Figures, unless otherwise noted,“Frz1-Fc, Frz2-Fc, Frz3-Fc, Frz4-Fc, Frz6-Fc, Frz7-Fc, Frz9-Fc,Frz-10-Fc, sFRP1-Fc, sFRP2-Fc, and sFRP4-Fc” refer to the respectivechimeras shown in FIGS. 7A, 7B and 7C (SEQ ID NOs: 76-85, and 87).

The Wnt antagonists are stable in vivo. Prior constructs utilizing aFrizzled domain attached to a Fc component were rapidly degraded in vivomaking them unsuitable for use as therapeutic compounds (Hsieh, J-C. etal., PNAS, 96: 3546-3551 (1999)). The Wnt antagonists described hereinremain stable in vivo for substantially longer than the priorconstructs. As shown in Example 4 (FIG. 13), the Frz8-Fc Wnt antagonistdisplayed an in vivo half-life of about 4 days. Accordingly, theinvention provides for Wnt antagonists that have an in vivo half-life ofat least 1 day, 2 days, 3 days, or 4 days after being administered to amammal.

Furthermore, as shown in Example 3 (FIG. 11), the Wnt antagonists retainactivity in vivo for substantially longer than the prior constructs. Inone embodiment, the Wnt antagonist is active for at least 30 minutes, 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30hours, 36 hours, 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60hours, 64 hours, 68 hours, 72 hours, 80 hours, 90 hours, or 100 hoursafter being administered to a mammal. Activity is measured, for example,by testing the serum of the mammal administered the Wnt antagonist forthe ability to inhibit Wnt signaling as set forth in Examples 3 and 11,or by using other methods known in the art.

2. Nucleic Acids

One aspect of the invention provides for a nucleic acid encoding the Wntantagonists described herein. In specific embodiments, the nucleic acidencodes a Wnt antagonist comprising a CRD (ECD)s of hFrz1 (SEQ ID NO:18), hFrz2 (SEQ ID NO: 19), hFrz3 (SEQ ID NO: 20), hFrz4 (SEQ ID NO:21), hFrz5 (SEQ ID NO: 22), hFrz6 (SEQ ID NO: 23), hFrz7 (SEQ ID NO:24), hFrz8 (SEQ ID NO: 25), hFrz9 (SEQ ID NO: 26), hFrz10 (SEQ ID NO:27), sFRP1 (SEQ ID NO: 28), sFRP2 (SEQ ID NO: 29), sFRP3 (SEQ ID NO:30), sFRP4 (SEQ ID NO: 31), sFRP5 (SEQ ID NO: 32), hRor1 (SEQ ID NO:33), or hRor2 (SEQ ID NO: 34).

In other embodiments, the nucleic acid encodes a Wnt antagonistcomprising a pro-Frz or pro-sFrp proteins selected from among hFrz1 (SEQID NO: 35), hFrz2 (SEQ ID NO: 36), hFrz3 (SEQ ID NO: 37), hFrz4 (SEQ IDNO: 38), hFrz5 (SEQ ID NO: 39), hFrz6 (SEQ ID NO: 40), hFrz7 (SEQ ID NO:41), hFrz8 (SEQ ID NO: 42), hFrz9 (SEQ ID NO: 43), hFrz10 (SEQ ID NO:44), sFRP1 (SEQ ID NO: 45), sFRP2 (SEQ ID NO: 46), sFRP3 (SEQ ID NO:47), sFRP4 (SEQ ID NO: 48), and sFRP5 (SEQ ID NO: 49).

In still other embodiments, the nucleic acid encodes a Wnt antagonistcomprising a mature Frz, sFRP or hRor proteins selected from among hFrz1(SEQ ID NO: 50), hFrz2 (SEQ ID NO: 51), hFrz3 (SEQ ID NO: 52), hFrz4(SEQ ID NO: 53), hFrz5 (SEQ ID NO: 54), hFrz6 (SEQ ID NO: 55), hFrz7(SEQ ID NO: 56), hFrz8 (SEQ ID NO: 57), hFrz9 (SEQ ID NO: 58), hFrz10(SEQ ID NO: 59), sFRP1 (SEQ ID NO: 60), sFRP2 (SEQ ID NO: 61), sFRP3(SEQ ID NO: 62), sFRP4 (SEQ ID NO: 63), sFRP5 (SEQ ID NO: 64), hRor1(SEQ ID NO: 65), and hRor2 (SEQ ID NO: 66).

In still other embodiments, the nucleic acid encodes a Wnt antagonistcomprising a Frz8-Fc (SEQ ID NO: 74), Frz5-Fc (SEQ ID NO: 75), Frz1-Fc(SEQ ID NO: 76), Frz2-Fc (SEQ ID NO: 77), Frz3-Fc (SEQ ID NO: 78),Frz4-Fc (SEQ ID NO: 79), Frz6-Fc (SEQ ID NO: 80), Frz7-Fc (SEQ ID NO:81), Frz9-Fc (SEQ ID NO: 82), Frz10-Fc (SEQ ID NO: 83), sFRP1-Fc (SEQ IDNO: 84), sFRP2 (SEQ ID NO: 85), sFRP3-Fc (SEQ ID NO: 86), sFRP4-Fc (SEQID NO: 87), or sFRP5-Fc (SEQ ID NO: 88).

In one particular embodiment, the nucleic acid encodes a Frz8-Fc andcomprises the nucleic acid sequence shown in SEQ ID NO: 122 (FIG. 5D).In another embodiment, the nucleic acid encodes a Frz5-Fc and comprisesthe nucleic acid sequence shown in SEQ ID NO: 119 (FIG. 5C). In yetfurther embodiments, the nucleic acid encodes a Frz1-Fc, Frz2-Fc,Frz3-Fc, Frz4-Fc, Frz6-Fc, Frz7-Fc, Frz9-Fc, Frz10-Fc, sFRP1-Fc, sFRP2,sFRP3-Fc, sFRP4-Fc, or sFRP5-Fc and comprises a nucleic acid sequenceshown in FIG. 5 (A-H). For example, the nucleic acid comprises a Frz1-Fc(SEQ ID NO: 115), Frz2-Fc (SEQ ID NO: 116), Frz3-Fc (SEQ ID NO: 117),Frz4-Fc (SEQ ID NO: 118), Frz5-Fc (SEQ ID NO: 119), Frz6-Fc (SEQ ID NO:120), Frz7-Fc (SEQ ID NO: 121), Frz8-Fc (SEQ ID NO: 122), Frz9-Fc (SEQID NO: 123), Frz10-Fc (SEQ ID NO: 124), sFRP1-Fc (SEQ ID NO: 125),sFRP2-Fc (SEQ ID NO: 126), sFRP3-Fc (SEQ ID NO: 127), sFRP4-Fc (SEQ IDNO: 128), or sFRP5-Fc (SEQ ID NO:129).

Another aspect of the invention provides for nucleic acids thathybridize under high stringency conditions to the nucleic acidsdescribed above.

3. Wnt Antagonist Variants

In addition to the Wnt antagonist polypeptides described herein, it iscontemplated that Wnt antagonist variants can be prepared. Such variantscan be prepared by introducing appropriate nucleotide changes into theencoding DNA, and/or by synthesis of the desired variant. Those skilledin the art will appreciate that amino acid changes may alterpost-translational processes of the Wnt antagonist, such as changing thenumber or position of glycosylation sites or altering the membraneanchoring characteristics.

A Wnt antagonist variant includes, for example, a mutation or amino acidvariant in an amino acid residue in one or more domains, while stillretaining biological activity. A Wnt antagonist variant also includesWnt antagonists having at least one amino acid deletion or addition,while still retaining biological activity. The addition or deletion ofthe amino acid residues can particularly occur in the region surroundingthe amino acid sequence where the Frz domain component and Fc domain areconnected, whether or not such region contains a linker. Wnt antagonistvariants have at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identitywith a reference Wnt antagonist polypeptide sequence. In general suchvariants exhibit substantially the same or greater binding affinity to aWnt protein than the reference sequence, e.g., at least 0.75×, 0.8×,0.9×, 1.0×, 1.25× or 1.5×, based on an art-accepted binding assayquantitation unit/metric.

In specific embodiments, the Wnt antagonist variant is a chimericmolecule comprising a Frz domain component having at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% amino acid sequence identity with the CRD (ECD)s of hFrz1(SEQ ID NO: 18), hFrz2 (SEQ ID NO: 19), hFrz3 (SEQ ID NO: 20), hFrz4(SEQ ID NO: 21), hFrz5 (SEQ ID NO: 22), hFrz6 (SEQ ID NO: 23), hFrz7(SEQ ID NO: 24), hFrz8 (SEQ ID NO: 25), hFrz9 (SEQ ID NO: 26), hFrz10(SEQ ID NO: 27), sFRP1 (SEQ ID NO: 28), sFRP2 (SEQ ID NO: 29), sFRP3(SEQ ID NO: 30), sFRP4 (SEQ ID NO: 31), sFRP5 (SEQ ID NO: 32), hRor1(SEQ ID NO: 33), or hRor2 (SEQ ID NO: 34).

In other embodiments, the Wnt antagonist variant is a chimeric moleculecomprising a Frz domain component having at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%amino acid sequence identity with a pro-Frz or pro-sFrp proteinsselected from among hFrz1 (SEQ ID NO: 35), hFrz2 (SEQ ID NO: 36), hFrz3(SEQ ID NO: 37), hFrz4 (SEQ ID NO: 38), hFrz5 (SEQ ID NO: 39), hFrz6(SEQ ID NO: 40), hFrz7 (SEQ ID NO: 41), hFrz8 (SEQ ID NO: 42), hFrz9(SEQ ID NO: 43), hFrz10 (SEQ ID NO: 44), sFRP1 (SEQ ID NO: 45), sFRP2(SEQ ID NO: 46), sFRP3 (SEQ ID NO: 47), sFRP4 (SEQ ID NO: 48), and sFRP5(SEQ ID NO: 49).

In still other embodiments, the Wnt antagonist variant is a chimericmolecule comprising a Frz domain component having at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% amino acid sequence identity with mature Frz, sFRP or hRorproteins selected from among hFrz1 (SEQ ID NO: 50), hFrz2 (SEQ ID NO:51), hFrz3 (SEQ ID NO: 52), hFrz4 (SEQ ID NO: 53), hFrz5 (SEQ ID NO:54), hFrz6 (SEQ ID NO: 55), hFrz7 (SEQ ID NO: 56), hFrz8 (SEQ ID NO:57), hFrz9 (SEQ ID NO: 58), hFrz10 (SEQ ID NO: 59), sFRP1 (SEQ ID NO:60), sFRP2 (SEQ ID NO: 61), sFRP3 (SEQ ID NO: 62), sFRP4 (SEQ ID NO:63), sFRP5 (SEQ ID NO: 64), hRor1 (SEQ ID NO: 65), and hRor2 (SEQ ID NO:66).

In still other embodiments, the Wnt antagonist variant is a chimericmolecule comprising a Frz domain component having at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% amino acid sequence identity with Frz8-Fc (SEQ ID NO: 74),Frz5-Fc (SEQ ID NO: 75), Frz1-Fc (SEQ ID NO: 76), Frz2-Fc (SEQ ID NO:77), Frz3-Fc (SEQ ID NO: 78), Frz4-Fc (SEQ ID NO: 79), Frz6-Fc (SEQ IDNO: 80), Frz7-Fc (SEQ ID NO: 81), Frz9-Fc (SEQ ID NO: 82), Frz10-Fc (SEQID NO: 83), sFRP1-Fc (SEQ ID NO: 84), sFRP2 (SEQ ID NO: 85), sFRP3-Fc(SEQ ID NO: 86), sFRP4-Fc (SEQ ID NO: 87), and sFRP5-Fc (SEQ ID NO: 88).

“Percent (%) amino acid sequence identity” is defined as the percentageof amino acid residues that are identical with amino acid residues in areference (parent) polypeptide sequence when the two sequences arealigned. To determine % amino acid identity, sequences are aligned andif necessary, gaps are introduced to achieve the maximum % sequenceidentity; conservative substitutions are not considered as part of thesequence identity. Amino acid sequence alignment procedures to determinepercent identity are well known to those of skill in the art. Oftenpublicly available computer software such as BLAST, BLAST2, ALIGN2 orMegalign (DNASTAR) software is used to align peptide sequences. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared.

When amino acid sequences are aligned, the % amino acid sequenceidentity of a given amino acid sequence A to, with, or against a givenamino acid sequence B (which can alternatively be phrased as a givenamino acid sequence A that has or comprises a certain % amino acidsequence identity to, with, or against a given amino acid sequence B)can be calculated as:

% amino acid sequence identity=X/Y×100

where

X is the number of amino acid residues scored as identical matches bythe sequence alignment program's or algorithm's alignment of A and B

and

Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length ofamino acid sequence B, the % amino acid sequence identity of A to B willnot equal the % amino acid sequence identity of B to A.

An “isolated” or “purified” peptide, polypeptide, protein orbiologically active fragment is separated and/or recovered from acomponent of its natural environment. Contaminant components includematerials that would typically interfere with diagnostic or therapeuticuses for the polypeptide, and may include enzymes, hormones, and otherproteinaceous or non-proteinaceous materials. Preparations havingpreferably less than 30% by dry weight of non-desired contaminatingmaterial (contaminants), preferably less than 20%, 10%, and preferablyless than 5% contaminants are considered to be substantially isolated.An isolated, recombinantly-produced peptide/polypeptide or biologicallyactive portion thereof is preferably substantially free of culturemedium, i.e., culture medium represents preferably less than 20%,preferably less than about 10%, and preferably less than about 5% of thevolume of a peptide/polypeptide preparation. Examples of contaminantsinclude cell debris, culture media, and substances used and producedduring in vitro synthesis of the peptide/polypeptide.

Variations in the Wnt antagonist described herein, can be made, forexample, using any of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the antibody or polypeptide that results ina change in the amino acid sequence as compared with the native sequenceantibody or polypeptide. Optionally the variation is by substitution ofat least one amino acid with any other amino acid in one or more of thedomains Wnt antagonist. Guidance in determining which amino acid residuemay be inserted, substituted or deleted without adversely affecting thedesired activity may be found by comparing the sequence of the Wntantagonist with that of homologous known protein molecules andminimizing the number of amino acid sequence changes made in regions ofhigh homology. Amino acid substitutions can be the result of replacingone amino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with a serine,i.e., conservative amino acid replacements. Insertions, deletions orsubstitutions may optionally be in the range of about 1 to 5 aminoacids. The variation allowed may be determined by systematically makinginsertions, deletions or substitutions of amino acids in the sequenceand testing the resulting variants for activity exhibited by thefull-length or mature native sequence.

Wnt antagonists may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating antibody or polypeptidefragments by enzymatic digestion, e.g., by treating the protein with anenzyme known to cleave proteins at sites defined by particular aminoacid residues, or by digesting the DNA with suitable restriction enzymesand isolating the desired fragment. Yet another suitable techniqueinvolves isolating and amplifying a DNA fragment encoding a desiredantibody or polypeptide fragment, by polymerase chain reaction (PCR).Oligonucleotides that define the desired termini of the DNA fragment areemployed at the 5′ and 3′ primers in the PCR.

In particular embodiments, conservative substitutions of interest areshown in Table A under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table A, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE A Original Preferred Residue Exemplary Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;asp, lys; arg gln Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn;glu asn Glu (E) asp; gln asp Gly (G) ala ala His (H) asn; gln; lys; argarg Ile (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine;ile; val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe;ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr;cys cys Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; serphe Val (V) ile; leu; met; phe; ala; norleucine leu

Substantial modifications in function or immunological identity of theWnt antagonist are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;(2) neutral hydrophilic: Cys, Ser, Thr; Asn; Gln(3) acidic: Asp, Glu;(4) basic: His, Lys, Arg;(5) residues that influence chain orientation: Gly, Pro; and(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the Wnt antagonists of the invention.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244:1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

Any cysteine residue not involved in maintaining the proper conformationof the Wnt antagonist may also be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the Wntantagonist to improve its stability (particularly where the antibody isan antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g., a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g., 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the Wnt antagonist and Wnt protein. Such contactresidues and neighboring residues are candidates for substitutionaccording to the techniques elaborated herein. Once such variants aregenerated, the panel of variants is subjected to screening as describedherein and antibodies with superior properties in one or more relevantassays may be selected for further development.

Covalent modifications of Wnt antagonists are included within the scopeof this invention. One type of covalent modification includes reactingtargeted amino acid residues of a Wnt antagonist with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues of the Wnt antagonist. Derivatizationwith bifunctional agents is useful, for instance, for crosslinking theWnt antagonist to a water-insoluble support matrix or surface for use inthe method for purifying Wnt antagonists. Commonly used crosslinkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the Wnt antagonist includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the Frz, Wnt or sFRP polypeptide domains of theWnt antagonist. “Altering the native glycosylation pattern” is definedas deleting one or more carbohydrate moieties found in native sequenceof the component domains (either by removing the underlyingglycosylation site or by deleting the glycosylation by chemical and/orenzymatic means), and/or adding one or more glycosylation sites that arenot present in the native sequence component domain. In addition, thephrase includes qualitative changes in the glycosylation of the nativeproteins, involving a change in the nature and proportions of thevarious carbohydrate moieties present.

Glycosylation of antibodies and other polypeptides is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the Wnt antagonist is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the sequence of the original (i.e., pre-variant) Wntantagonist. This sequence may optionally be altered through changes atthe DNA level, particularly by mutating the DNA encoding the sequence atpreselected bases such that codons are generated that will translateinto the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theWnt antagonist is by chemical or enzymatic coupling of glycosides to thepolypeptide. Such methods are described in the art, e.g., in WO 87/05330published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev.Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the Wnt antagonist may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of Wnt antagonist compriseslinking the sequence to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The antibody orpolypeptide also may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington: The Science and Practice ofPharmacy, 20th edition, Gennaro, A., Ed., (2000).

The Wnt antagonists of the present invention may also be modified in away to form molecules having additional chimeric nature, comprising aWnt antagonist (i.e., Frz-, sFRP- or Ror-Fc chimera) fused to another,heterologous polypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of theWnt antagonist with a tag polypeptide which provides an epitope to whichan anti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the Wnt antagonist. Thepresence of such epitope-tagged forms of the Wnt antagonist can bedetected using an antibody against the tag polypeptide. Also, provisionof the epitope tag enables the Wnt antagonist to be readily purified byaffinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. Various tag polypeptidesand their respective antibodies are well known in the art. Examplesinclude poly-histidine (poly-his) or poly-histidine-glycine(poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5[Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag andthe 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.,Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al.,Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptidesinclude the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210(1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194(1992)]; an α-tubulin epitope peptide [Skinner et al., J. Biol. Chem.,266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag[Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397(1990)].

In alternative embodiments, the Wnt antagonists comprise a variant Fccomponent. For example, the Fc region may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g, a substitution) at one or more aminoacid positions including that of a hinge cysteine. In one embodiment,such variants have at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequenceidentity with a reference Fc polypeptide sequence.

In one embodiment, the Fc region variant may display altered neonatal Fcreceptor (FcRn) binding affinity. Such variant Fc regions may comprisean amino acid modification oat any one or more of amino acid positions238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400,413, 415, 424, 433, 434, 435, 436, 439 or 447 of the Fc region, whereinthe numbering of the residues in the Fc region is that of the EU indexas in Kabat. Fc region variants with reduced binding to an FcRn maycomprise an amino acid modification at any one or more of amino acidpositions 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435,436, 439 or 447 of Fc region (EU index/Kabat numbering). Alternatively,variants displaying increased binding to FcRn may comprise an amino acidmodification at any one or more of amino acid positions 238, 256, 265,272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378,380, 382, 413, 424 or 434 of the Fc region (EU index/Kabat numbering).

In another embodiment, the Fc region variant may display reduced bindingto an FcγR, and comprises amino acid modifications at positions 238,239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293,294, 295, 296, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340,373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438 or 439 of theFc region (EU index/Kabat numbering).

In yet another embodiment, the Fc region variant may display reducedbinding to FcγRII and comprises amino acid modifications at any one ormore of amino acid positions 238, 265, 269, 270, 292, 294, 295, 298,303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or439 of the Fc region (EU index/Kabat numbering).

In a further embodiment, the Fc region variant may display enhancedbinding to FcγRII, and comprises an amino acid modification at any oneor more of amino acid positions 238, 265, 269, 270, 292, 294, 295, 298,303, 324, 327, 329, 333, 338, 373, 376, 414, 416, 419, 435, 438 or 439of the Fc region (EU index/Kabat numbering).

In a still further embodiment, the Fc region variant of interest maydisplay reduced binding to an FcgRIII, and comprises an amino acidmodification at one or more amino acid positions 238, 239, 249, 252,254, 265, 268, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303,322, 327, 329, 338, 340, 373, 376, 382, 388, 389, 416, 434, 435 or 437of the Fc region (EU index/Kabat numbering).

In a still further embodiment, Fc region variants with altered (i.e,improved or diminished) C1q binding and/or complement dependentcytotoxicity (CDC) are described in WO99/51642. Such variants maycomprise an amino acid substitution at one or more of amino acidpositions 270, 322, 326, 327, 329, 331, 333 or 334 of the Fc region. Seealso, Duncan and Winter, Nature 322: 738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821 and WO94/29351 concerning Fc regionvariants.

B. Preparation of Wnt Antagonists

The description below relates primarily to production of Wnt antagonistpolypeptides by culturing cells transformed or transfected with a vectorcontaining Wnt antagonist polypeptide-encoding nucleic acid. It is, ofcourse, contemplated that alternative methods, which are well known inthe art, may be employed to prepare such Wnt antagonists. For instance,the appropriate amino acid sequence, or portions thereof, may beproduced by direct peptide synthesis using solid-phase techniques [see,e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co.,San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc.,85:2149-2154 (1963)]. In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may beaccomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturer's instructions.

Various portions of the Wnt antagonist polypeptide may be chemicallysynthesized separately and combined using chemical or enzymatic methodsto produce the desired sequence.

1. Isolation of DNA Encoding Wnt Antagonist Polypeptide

DNA encoding the sequence of the antagonists or any desired componentdomains of the Wnt antagonist, such as an Frz, or sFRP may be obtainedfrom a cDNA library prepared from tissue believed to possess suchsequence and to express it at a detectable level. Accordingly, a humanFrz or sFRP sequence DNA can be conveniently obtained from a cDNAlibrary prepared from human tissue. The desired DNA sequence gene mayalso be obtained from a genomic library or by known synthetic procedures(e.g., automated nucleic acid synthesis).

Libraries can be screened with probes (such as oligonucleotides of atleast about 20-80 bases) designed to identify the gene of interest orthe protein encoded by it. Screening the cDNA or genomic library withthe selected probe may be conducted using standard procedures, such asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual(New York: Cold Spring Harbor Laboratory Press, 1989). An alternativemeans to isolate the gene encoding Wnt antagonist polypeptide andcomponents thereof is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

Techniques for screening a cDNA library are well known in the art. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

DNA sequence encoding Fc immunoglobulin domains may be derived fromhybridoma cells secreting mAbs of the desired Fc subtype.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for Wnt antagonist polypeptide production andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. The culture conditions, such as media,temperature, pH and the like, can be selected by the skilled artisanwithout undue experimentation. In general, principles, protocols, andpractical techniques for maximizing the productivity of cell culturescan be found in Mammalian Cell Biotechnology: A Practical Approach, M.Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

Full length antibody, antibody fragments, and antibody fusion proteinscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in tumor cell destruction.Full length antibodies have greater half life in circulation. Productionin E. coli is faster and more cost efficient. For expression of antibodyfragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), andU.S. Pat. No. 5,840,523 (Simmons et al.) which describes translationinitiation regio (TIR) and signal sequences for optimizing expressionand secretion, these patents are incorporated herein by reference. Afterexpression, the antibody is isolated from the E. coli cell paste in asoluble fraction and can be purified through, e.g., a protein A or Gcolumn depending on the isotype. Final purification can be carried outsimilar to the process for purifying antibody expressed e.g, in CHOcells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for Wntantagonist polypeptide-encoding vectors. Saccharomyces cerevisiae is acommonly used lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al.,J. Basic Microbiol., 28:265-278 [1988]); Candida; Trichoderma reesia (EP244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis(EP 394,538 published 31 Oct. 1990); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al.,Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al.,Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated Wnt antagonistpolypeptide are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells such as Drosophila S2 andSpodoptera Sf9, as well as plant cells, such as cell cultures of cotton,corn, potato, soybean, petunia, tomato, and tobacco. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for Wnt antagonist polypeptide production and culturedin conventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

3. Selection and Use of a Replicable Vector

One aspect of the invention provides for the nucleic acid (e.g., cDNA orgenomic DNA) encoding a Wnt antagonist polypeptide inserted into areplicable vector for cloning (amplification of the DNA) or forexpression. Various vectors are publicly available. The vector may, forexample, be in the form of a plasmid, cosmid, viral particle, or phage.The appropriate nucleic acid sequence may be inserted into the vector bya variety of procedures. In general, DNA is inserted into an appropriaterestriction endonuclease site(s) using techniques known in the art.Vector components generally include, but are not limited to, one or moreof a signal sequence, an origin of replication, one or more markergenes, an enhancer element, a promoter, and a transcription terminationsequence. Construction of suitable vectors containing one or more ofthese components employs standard ligation techniques which are known tothe skilled artisan.

The Wnt antagonist may be produced recombinantly not only directly, butalso as a fusion polypeptide with a heterologous polypeptide, which maybe a signal sequence or other polypeptide having a specific cleavagesite at the N-terminus of the mature protein or polypeptide. In general,the signal sequence may be a component of the vector, or it may be apart of the Wnt antagonist polypeptide-encoding DNA that is insertedinto the vector. The signal sequence may be a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up the Wntantagonist-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the Wnt antagonist-encoding nucleic acid sequence to directmRNA synthesis. Promoters recognized by a variety of potential hostcells are well known. Promoters suitable for use with prokaryotic hostsinclude the β-lactamase and lactose promoter systems [Chang et al.,Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)]. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theWnt antagonist polypeptide.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

Wnt antagonist polypeptide transcription from vectors in mammalian hostcells is controlled, for example, by promoters obtained from the genomesof viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter, and from heat-shockpromoters, provided such promoters are compatible with the host cellsystems.

Transcription of a DNA encoding the Wnt antagonist polypeptide by highereukaryotes may be increased by inserting an enhancer sequence into thevector Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, α-fetoprotein, and insulin). Typically, however, one will usean enhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theWnt antagonist polypeptide coding sequence, but is preferably located ata site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding Wnt antagonist.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of Wnt antagonist polypeptide in recombinant vertebratecell culture are described in Gething et al., Nature, 293:620-625(1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP117,058.

4. Culturing the Host Cells

One aspect of the invention provides for a host cell comprising thenucleic acid encoding the Wnt antagonists. The host cells used toproduce the Wnt antagonist polypeptide of this invention may be culturedin a variety of media. Commercially available media such as Ham's F10(Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma),and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be usedas culture media for the host cells. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas GENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

5. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a Frz, sFRP or Rorsequence identified herein or against a synthetic peptide based on theDNA sequences provided herein or against exogenous sequence fused to theWnt antagonist and encoding a specific antibody epitope.

6. Purification of Wnt Antagonist

Forms of Wnt antagonist polypeptide may be recovered from culture mediumor from host cell lysates. If membrane-bound, it can be released fromthe membrane using a suitable detergent solution (e.g. Triton-X 100) orby enzymatic cleavage. Cells employed in expression of Wnt antagonistpolypeptide can be disrupted by various physical or chemical means, suchas freeze-thaw cycling, sonication, mechanical disruption, or celllysing agents.

It may be desired to purify Wnt antagonist polypeptide from recombinantcell proteins or polypeptides. The following procedures are exemplary ofsuitable purification procedures: by fractionation on an ion-exchangecolumn; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation-exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; protein A Sepharose columns to removecontaminants such as IgG; and metal chelating columns to bindepitope-tagged forms of the Wnt antagonist. Various methods of proteinpurification may be employed and such methods are known in the art anddescribed for example in Deutscher, Methods in Enzymology, 182 (1990);Scopes, Protein Purification: Principles and Practice, Springer-Verlag,New York (1982). The purification step(s) selected will depend, forexample, on the nature of the production process used and the particularWnt antagonist polypeptide produced.

When using recombinant techniques, the Wnt antagonist polypeptide can beproduced intracellularly, in the periplasmic space, or directly secretedinto the medium. If the Wnt antagonist polypeptide is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, are removed, for example, by centrifugation orultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992)describe a procedure for isolating antibodies which are secreted to theperiplasmic space of E. coli. Briefly, cell paste is thawed in thepresence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris canbe removed by centrifugation. Where the Wnt antagonist polypeptide issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The Wnt antagonist polypeptide composition prepared from the cells canbe purified using, for example, hydroxylapatite chromatography, gelelectrophoresis, dialysis, and affinity chromatography, with affinitychromatography being the preferred purification technique. Thesuitability of protein A as an affinity ligand depends on the speciesand isotype of any immunoglobulin Fc domain that is present in theantibody. Protein A can be used to purify antibodies that are based onhuman γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe Wnt antagonist polypeptide comprises a C_(H)3 domain, the BakerbondABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.Other techniques for protein purification such as fractionation on anion-exchange column, ethanol precipitation, Reverse Phase HPLC,chromatography on silica, chromatography on heparin SEPHAROSE™chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the antibody to berecovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

C. Pharmaceutical Formulations

One aspect of the invention provides for a composition comprising a Wntantagonist and at least one pharmaceutically acceptable carrier orexcipient. Therapeutic formulations of the Wnt antagonists used inaccordance with the present invention are prepared for storage by mixingthe Wnt antagonists having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington: The Science and Practice of Pharmacy, 20th edition, A.Gennaro, Ed. (2000)), in the form of lyophilized formulations or aqueoussolutions. A “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Additional examples ofsuitable carriers or diluents include, but are not limited to, water,saline, Finger's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. Except when a conventional media or agent is incompatible withan active compound, use of these compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as acetate, Tris, phosphate, citrate, and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA;tonicifiers such as trehalose and sodium chloride; sugars such assucrose, mannitol, trehalose or sorbitol; surfactant such aspolysorbate; salt-forming counter-ions such as sodium; metal complexes(e.g., Zn-protein complexes); and/or non-ionic surfactants such asTWEEN®, PLURONICS® or polyethylene glycol (PEG). The antibody preferablycomprises the antibody at a concentration of between 5-200 mg/ml,preferably between 10-100 mg/ml.

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, in addition to a particular Wnt antagonist, it maybe desirable to include in the one formulation, an additional antibody,e.g., which binds a different epitope on the Wnt protein, to a differentWnt protein entirely, or an antibody to some other target such as agrowth factor that affects the growth of the Wnt mediated disorder.Alternatively, or additionally, the composition may further comprise achemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitoryagent, anti-hormonal agent, and/or cardioprotectant. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington: The Science and Practice of Pharmacy, 20th edition, A.Gennaro, Ed. (2000).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics”, InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.

When in vivo administration of a substance or molecule of the inventionis employed, normal dosage amounts may vary from about 10 ng/kg to up to100 mg/kg of mammal body weight or more per day, preferably about 1μg/kg/day to 10 mg/kg/day, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344;or 5,225,212. It is anticipated that different formulations will beeffective for different treatment compounds and different disorders,that administration targeting one organ or tissue, for example, maynecessitate delivery in a manner different from that to another organ ortissue.

Where sustained-release administration of a substance or molecule isdesired in a formulation with release characteristics suitable for thetreatment of any disease or disorder requiring administration of thesubstance or molecule, microencapsulation of the substance or moleculeis contemplated. Suitable examples of sustained-release preparationsinclude semi-permeable matrices of solid hydrophobic polymers containingthe antibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Microencapsulation of recombinant proteins forsustained release has been successfully performed with human growthhormone (rhGH), interferon-α,γ (rhIFN-α,-γ), interleukin-2, and MNrgp120. Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda, Biomed.Ther., 27:1221-1223 (1993); Hora et al., Bio/Technology, 8:755-758(1990); Cleland, “Design and Production of Single Immunization VaccinesUsing Polylactide Polyglycolide Microsphere Systems,” in Vaccine Design:The Subunit and Adjuvant Approach, Powell and Newman, eds, (PlenumPress: New York, 1995), pp. 439-462; WO 97/03692, WO 96/40072, WO96/07399; and U.S. Pat. No. 5,654,010.

The sustained-release formulations of these proteins may be developedusing poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1-41.

Additional examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT7 (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid.

D. Methods of Treating Wnt Mediated Disorder

The invention provides for methods of treating a Wnt-mediated disorderin a mammal suffering therefrom, comprising administering to the mammala therapeutically effective amount of a Wnt antagonist. In oneembodiment, the disorder is a cell proliferative disorder associatedwith aberrant, e.g., increased, expression of activity of Wnt signaling.In another embodiment, the disorder results from increased expression ofa Wnt protein. In yet another embodiment, the cell proliferativedisorder is cancer, such as for example, colon cancer, colorectalcancer, breast cancer, cancer associated with various disorders relatingto HSC's, such as leukemias and various other blood related cancers, andcancer related to neuronal proliferative disorders, including braintumors, such as gliomas, astrocytomas, meningiomas, Schwannomas,pituitary tumors, primitive neuroectodermal tumors (PNET),medulloblastomas, craniopharyngioma, and pineal region tumors.

Treatment of the cell proliferative disorder by administration of a Wntantagonist results in an observable and/or measurable reduction in orabsence of one or more of the following: reduction in the number ofcancer cells or absence of the cancer cells; reduction in the tumorsize; inhibition (i.e., slow to some extent and preferably stop) ofcancer cell infiltration into peripheral organs including the spread ofcancer into soft tissue and bone; inhibition (i.e., slow to some extentand preferably stop) of tumor metastasis; inhibition, to some extent, oftumor growth; and/or relief to some extent, one or more of the symptomsassociated with the specific cancer; reduced morbidity and mortality,and improvement in quality of life issues. To the extent the Wntantagonist may prevent growth and/or kill existing cancer cells, it maybe cytostatic and/or cytotoxic. Reduction of these signs or symptoms mayalso be felt by the patient.

The above parameters for assessing successful treatment and improvementin the disease are readily measurable by routine procedures familiar toa physician. For cancer therapy, efficacy can be measured, for example,by assessing the time to disease progression (TDP) and/or determiningthe response rate (RR). Metastasis can be determined by staging testsand by bone scan and tests for calcium level and other enzymes todetermine spread to the bone. CT scans can also be done to look forspread to the pelvis and lymph nodes in the area. Chest X-rays andmeasurement of liver enzyme levels by known methods are used to look formetastasis to the lungs and liver, respectively. Other routine methodsfor monitoring the disease include transrectal ultrasonography (TRUS)and transrectal needle biopsy (TRNB).

In a specific embodiment, the administration of Wnt antagonist decreasestumor burden (e.g., reduces size or severity of the cancer). In yetanother specific embodiment, the administration of Wnt antagonist killsthe cancer.

E. Methods of Inhibiting Wnt-Signaling in a Cell

The invention provides for a method of inhibiting Wnt-signaling in acell comprising contacting the cell with an effective amount of a Wntantagonist. In one embodiment, the cell is contained within a mammal,preferably a human, and the administered amount is a therapeuticallyeffective amount. In yet another embodiment, the inhibition of Wntsignaling further results in the inhibition of the growth of the cell.In a further embodiment, the cell is a cancer cell.

Inhibition of cell proliferation is measured using methods known tothose skilled in the art. For example, a convenient assay for measuringcell proliferation is the CellTiter-Glo™ Luminescent Cell ViabilityAssay, which is commercially available from Promega (Madison, Wis.).That assay determines the number of viable cells in culture based onquantitation of ATP present, which is an indication of metabolicallyactive cells. See Crouch et al (1993) J. Immunol. Meth. 160:81-88, U.S.Pat. No. 6,602,677. The assay may be conducted in 96- or 384-wellformat, making it amenable to automated high-throughput screening (HTS).See Cree et al (1995) AntiCancer Drugs 6:398-404. The assay procedureinvolves adding a single reagent (CellTiter-Glo® Reagent) directly tocultured cells. This results in cell lysis and generation of aluminescent signal produced by a luciferase reaction. The luminescentsignal is proportional to the amount of ATP present, which is directlyproportional to the number of viable cells present in culture. Data canbe recorded by luminometer or CCD camera imaging device. Theluminescence output is expressed as relative light units (RLU).

F. Methods of Modulating the Expression of a Wnt Target Gene

The invention provides for a method of modulating the expression of aWnt target gene in a cell characterized by activated or excessive Wntsignaling, comprising contacting the cell with an effective amount of aWnt antagonist. In one embodiment, the Wnt target gene is overexpressedas a result of the Wnt signaling, and the result of the contact with theWnt antagonist reduces expression of the Wnt target gene. In anotherembodiment, the Wnt target gene is selected from the group consistingof: Axin2, APCDD1, Gad1, Sax1, c-myc, cyclin D1, PPARdelta, gastrin,clusterin, survivin, cyclooxygenase, fra-1, osteopontin, uPAR,claudin-1, CD44, MMP-7/9/11/14/26, IGFBP-4, Met, BMP4, sox-9, histonedeacetylase 2, VEGF. In yet another embodiment, the Wnt target gene isunderexpressed as a result of the Wnt signaling, and the result ofcontact with the Wnt antagonist restores expression of the Wnt targetgene. In a further embodiment, the Wnt target gene is selected from thegroup consisting of: Lefty′, Lefty2, sFRP1, Fzd5, fas antigen, caspase3, integrin β7, alpha e integrin, hath 1, fatty acid binding protein 2,muc-2, kruppel like factor-4, carbonic anhydrase-11, EphrinB1, EphB2R,EphB3R, muc-3, histocompatibility 2, Q region locus 1, β2-microglobulin.

Expression of the target genes is determined using methods known tothose of skill in the art, including those described herein and setforth in the Examples below.

G. Methods of Detecting the Presence of a Wnt Protein

The invention provides for a method of detecting the presence of a Wntprotein in a sample, comprising contacting the sample with a Wntantagonist, wherein the presence of a complex or the level of bindingbetween the Wnt antagonist and the Wnt protein is indicative of thepresence of Wnt protein and/or Wnt signaling. In one embodiment, themethod further comprises determining if the level of Wnt signaling isaberrant. In this embodiment, the level of Wnt protein binding in thesample is compared with the level in a second sample in which Wntprotein expression and/or Wnt signaling is known to be physiologicallynormal. The level of binding in the suspect sample compared to thesecond sample that is higher or lower than the physiologically normalsample is indicative of aberrant Wnt signaling. In another embodiment,the presence of Wnt signaling or aberrant Wnt signaling is indicative ofthe presence of a Wnt-mediated disorder, such as cancer.

H. The Wnt Pathway and Disorders Associated Therewith 1. The WntSignaling Pathway:

The Wnt signaling pathway is an unusually complex signaling processinvolving multiple proteins which exert varying levels of control in thepathway. This multi-level, tight regulation of the pathway is indicativeof its importance in cellular biology. Despite the complicatedregulatory mechanisms, the initial signal of the pathway is generated bythe binding of a Wnt to the Frizzled (Frz) receptors. Effective signalfurther requires the presence of an additional single pass transmembranemolecule of the LRP (LDL receptor related protein) class, specificallyLRP 5 and LRP 6. Wnt may further bind with LRP to form a trimericcomplex with Frizzled. The cytoplasmic tail of LRP in turn interactswith Axin, another downstream component. Dishevelled, a cytoplasmiccomponent that interacts directly with Frizzled, may also directlyinteract with Axin, thus forming a tetra-plex complex of Frizzled, LRP,Dsh and Axin. This interaction with Axin releases β-catenin from the“degradation complex” (discussed infra) for subsequent downstreamactivity in the Wnt signaling pathway.

Outside the cell, Wnt signaling is inhibited by various proteins thatcan bind to Wnt thereby sequestering it from its receptor. Included inthis group are the secreted Frizzled related proteins (sFRPs, Jones etal., Bioessays 2002; 24: 811-820) and Wnt inhibitory factor-1 (WIF-1,Hsieh, J. C. et al., Nature 1999; 398: 431-436). In humans, the sFRPfamily consists of five members (e.g., sFRP-1, sFRP-2 . . . sFRP-5),each containing a cysteine-rich domain (CRD) which shares 30-50%sequence homology with the CRD of Frz receptors. (Melkonyan, H. S. etal., Proc. Natl. Acad. Sci. USA 1997; 94: 13636-13641). sFRPs arebelieved to form function-inhibiting complexes with Frz receptors, andtherefore are natural antagonists, but the biology is complex, and insome cases, may even act to agonize Wnt activity. (Uren, A. et al., J.Biol. Chem. 2000; 275: 4374-4382).

Another class of extracellular Wnt inhibitor is Dickkopf (Dkk). [Brott,B. K. et al., Mol. Cell Biol. 2002; 22: 6100-6110; Fedi, P. et al., J.Biol. Chem. 1999; 274: 19465-19472] The three members of the Dkk family(e.g., Dkk-1, Dkk-2 and Dkk-4) can antagonize Wnt signaling throughinactivation of the cell surface receptor LRP-5 and LRP-6, essentialcomponents of the canonical pathway. [Mao, J. H. et al., Mol. Cell 2001;7: 801-809; Pinson, K. I. et al., Nature 2000; 407: 535-538]. Dkk formsa ternary complex with LRP5/6 and the single pass transmembranereceptors Kremen 1 (Krm-1) or Kremen 2 (Krm-2) [Mao et al., Gene 2003;302: 179-183; Mao et al., Nature 2002; 417: 664-667; Mao et al., Nature2001; 411: 321-325]. This complex in turn undergoes endocytosis, therebyremoving LRP5/6 receptors from the cell surface. As a result, Dkks canselectively antagonize canonical Wnt signaling, while not affectingnon-canonical signaling.

The hallmark of canonical Wnt signaling activation is elevated levels ofthe protein β-catenin. β-catenin is constitutively produced and ispresent in the cytoplasm as pools of monomeric protein. [Papkoff, J. etal., Mol. Cell Biol. 1996; 16: 2128-2134]. The primary mechanism forcontrolling cytoplasmic levels of β-catenin is through direct physicaldegradation upon recruitment into a large multi-protein complex(“degradation complex”). The central scaffolding of this complex isprovided by Axin, as well as binding sites for β-catenin, adenomatouspolyposis coli (APC), glycogen synthase kinase 3β (GSK3(3), caseinkinase Iα (CKIα) and protein phophatase 2A (PP2A) [Hinoi, T. et al., J.Biol. Chem. 2000; 275: 34399-34406; Ikeda et al., Oncogene 2000; 19:537-545; Yamamoto et al., J. Biol. Chem. 1999; 274: 10681-10684; Kishidaet al., J. Biol. Chem. 1998; 273: 10823-10826; Ikeda et al., EMBO 11998; 17: 1371-1384. After formation, the complex is stabilized by theGSK3β-mediated phosphorylation of Axin and APC, as well as PP2A. GSK3β-then phosphorylates β-catenin thereby allowing it be recognized byβ-transducin repeat containing protein (β-TrCP), thereby targeting itfor ubiquitination and proteosomic degradation. [Aberle et al., EMBO J.1997; 16: 3797-804; Latres et al., Oncogene 1999; 18: 849-54; Liu etal., Proc. Natl. Acad. Sci. USA 1999; 96: 6273-8].

Although complexation with Axin/APC/GSK3β is the primary mechanism fordegradation of β-catenin, an alternative degradation pathway has beenshown involving ubiquitination induced by complexation with Siah-1 andthe C-terminus of APC. [Matsuzawa et al., Mol. Cell 2001; 7: 915-926;Liu et al., Mol. Cell 2001; 7: 927-936]. In addition to its role as atranscription factor, β-catenin further is involved in cellularadhesion. [Nelson et al., Science 2004; 303: 1483-1487; Ilyas et al., J.Pathol. 1997; 182: 128-137. β-catenin can be found at the cell surfacesites of intercellular contact known as adherens junctions, where it iscomplexed with E-cadherin and α-catenin. Thus, any increase inE-cadherin expression will direct β-catenin to the cell membrane,thereby depleting cytoplasmic levels, and in turn inhibit Wnt signaling.Moreover, the breakdown of the E-cadherin-catenin complex can increasecytoplasmic levels of free β-catenin, thereby stimulatingtranscriptional activity. [Nelson et al., supra]. Thus, activation ofthe cell surface receptors cRON, epidermal growth factor receptor (EGFR)and c-ErbB2, by liberating β-catenin, can also stimulate canonical Wntsignaling. Other signaling pathways that can either activate orfacilitate the effects of Wnt signaling. For example, integrin signalingcan result in nuclear transportation of β-catenin [Eger et al., Oncogene2004; 23: 2672-2680], while signaling through insulin-like growth factor(IGF) can activate Wnt signaling by “soaking up” available GSK3β—therebypreventing formation of the “degradation complex.”

In canonical signaling, an initial step involves the binding of Wnt toFrz in the presence of LRP5/6. [Mao et al., Mol. Cell 2001; 7: 801-809;Pinson et al., Nature 2000; 407: 535-538]. The formation of thistrimeric complex has two downstream consequences. First is therecruitment of Dishevelled (Dsh) to the cell surface and itsphosphorylation by casein kinase Iε (CIε) [Kishida et al., J. Biol.Chem. 2001; 276: 33147-33155]. The phosphorylated Dsh can form a complexwith Frat 1 and GSK3β, which in turn can inhibit the activity of GSK3β.Second, the Wnt/Frz/LRP5/6 tri-plex facilitates the LRP5/6 mediateddegradation of Axin. The net effect of this is the destabilization ofthe degradation complex responsible for phosphorylating β-catenin. Inthe absence of phosphorylation, β-catenin is not ubiquinated, therebyescaping degradation, thus increasing intracellular levels andavailability for translocation to the nucleus.

The manner in which β-catenin is transported to the nucleus is notentirely clear, but interaction with the nuclear transport proteins APC[Rosin-Arbesfeld et al., Nature 2000; 406: 1009-1012; Neufeld et al.,Proc. Natl. Acad. Sci. USA 2000; 97: 12085-12090], as well as pygopusand Bcl9/legless have been implicated. [Townsley et al., Nature CellBiol. 2004; 6: 626-633].

Once in the nucleus, β-catenin displaces the transcriptional repressorGroucho for binding with T-cell-specific transcription factor/lymphoidenhancer-binding factor-1 (TCF/LEF) DNA binding proteins. In the absenceof displacement by β-catenin, TCF/LEF complexes with Groucho to repressexpression of the Wnt “target genes”. The inhibitory effect of Grouchois further mediated by interactions with various histone deacetylases(HDAC), which are believed to make DNA refractive to transcriptionalactivation. [Cavallo et al., Nature 1998; 395: 604-8; Chen et al., GenesDev. 1999; 13: 2218-30]. The conversion of the TCF transcriptionalrepressor complex into a transcriptional activation complex furtherinvolves recruitment of histone acetylases such as Creb binding protein(CBP)/p300 as well as other activating factors such as Brg-1. [Takemaruet al., J. Cell Biol. 2000; 149: 249-54; Barker et al., Cell 2002; 109:47-60; Brantjes et al., Biol. Chem. 2002; 383: 255-261; Roose et al.,Biochim. Biophys Acta—Rev. Cancer 1999; 1424: M23-M37]. The interactionsbetween the β-catenin-TCF complex and chromatin also may be mediated byLegless (Bcl9) and Pygopus. Kramps et al., Cell 2002; 109: 47-60;Thompson et al., Nat. Cell Biol. 2002; 4: 367-73; Parker et al.,Development 2002; 129: 2565-76.

An abbreviated summary of the canonical Wnt signaling pathway both inthe “off” or inactive state as well as the “on” or active state isdepicted in FIG. 1.

2. Disorders Associated with Wnt Signaling Activity:

Deregulation of the Wnt signaling pathway may be caused by somaticmutations in genes encoding various Wnt signaling pathway components.For example, aberrant Wnt signaling activity has been associated withWnt ligand overexpression in non small cell lung cancer (NSCLC) [You etal., Oncogene 2004; 23: 6170-6174], chronic lymphocytic leukemia(CLL)[Lu et al., Proc. Natl. Acad. Sci. USA 2004; 101: 3118-3123],gastric cancer [Kim et al., Exp. Oncol. 2003; 25: 211-215; Saitoh etal., Int. J. Mol. Med. 2002; 9: 515-519], head and neck squamous cellcarcinoma (HNSCC) [Rhee et al., Oncogene 2002; 21: 6598-6605],colorectal cancer [Holcombe et al., J. Clin. Pathol—Mol. Pathol. 2002;55: 220-226], ovarian cancer [Ricken et al., Endocrinology 2002; 143:2741-2749], basal cell carcinoma (BCC) [Lo Muzio et al., Anticancer Res.2002; 22: 565-576] and breast cancer. Moreover, the reduction of variousWnt ligand regulatory molecules such as sFRP and WIF-1 have beenassociated with breast cancer [Klopocki et al., Int. I Oncol. 2004; 25:641-649; Ugolini et al., Oncogene 2001; 20: 5810-5817; Wissmann et al.,J. Pathol. 2003; 201: 204-212], bladder cancer [Stoehr et al., LabInvest. 2004; 84: 465-478; Wissmann et al., supra], mesothelioma [Lee etal., Oncogene 2004; 23: 6672-6676], colorectal cancer [Suzuki et al.,Nature Genet 2004; 36: 417-422; Kim et al., Mol. Cancer Ther. 2002; 1:1355-1359; Caldwell et al., Cancer Res. 2004; 64: 883-888], prostatecancer [Wissman et al., supra], NSCLC [Mazieres et al., Cancer Res.2004; 64: 4717-4720], and lung cancer [Wissman et al., supra].Antagonizing Wnt signaling with the Wnt antagonist molecules of theinvention is expected to therapeutically treat these cancers.

Continuing, aberrant Wnt signaling resulting from overexpression ofvarious components of the Frz-LRP receptor complex have also beenassociated with certain cancers. For example, LRP5 overexpression hasbeen associated with osteosarcoma [Hoang et al., Int. J Cancer 2004;109: 106-111], while Frz overexpression has been associated with cancerssuch as prostate [Wissmann et al., supra], HNSCC [Rhee et al., Oncogene2002; 21: 6598-6605], colorectal [Holcombe et al., supra], ovariancancer [Wissman et al, supra], esophageal [Tanaka et al., Proc. Natl.Acad. Sci. USA 1998; 95: 10164-10169] and gastric [Kirikoshi et al.,Int. J Oncol. 2001; 19: 111-115]. Additionally, overexpression of Wntsignaling pathway components such as Dishevelled have been associatedwith cancers such as prostate [Wissman et al., supra], breast [Nagahataet al., Cancer Sci. 2003; 94: 515-518], mesothelioma [Uematsu et al.,Cancer Res. 2003; 63: 4547-4551] and cervical [Okino et al., Oncol. Rep.2003; 10: 1219-1223]. Frat-1 overexpression has been associated withcancers such as pancreatic, esophageal, cervical, breast and gastric.[Saitoh et al., Int. J Oncol. 2002; 20: 785-789; Saitoh et al., Intl.Oncol. 2001; 19: 311-315]. Axin loss of function (LOF) mutations havebeen associated with hepatocellular cancer [Satoh et al., Nature Genet.2000; 24: 245-250; Taniguchi et al., Oncogene 2002; 21: 4863-4871] andmedulloblastoma [Dahmen et al., Cancer Res. 2001; 61: 7039-7043; Yokotaet al., Int. J Cancer 2002; 101: 198-201]. The blocking of Wnt-Frzinteractions with the Wnt antagonists of the present invention isexpected to alleviate cancers associated with overexpression of Frz orLRPs.

Finally, a multitude of cancers has been associated with activatingβ-catenin through disruption of the “degradation complex” such asgain-of-function mutations in β-catenin or loss-of-function mutations inAPC. A reduction in the degradation of β-catenin results in greateramounts of functional β-catenin in the cell, which then causes increasedtranscription of the target genes, resulting in aberrant cellproliferation. For example, mutations in the gene encoding β-catenin(i.e., CTNNB1) have been associated with cancers such as gastric[Clements et al., Cancer Res. 2002; 62: 3503-3506; Park et al., CancerRes. 1999; 59: 4257-4260], colorectal [Morin et al., Science 1997; 275:1787-1790; Ilyas et al., Proc. Natl Acad. Sci. USA 1997; 94:10330-10334], intestinal carcinoid [Fujimori et al., Cancer Res. 2001;61: 6656-6659], ovarian [Sunaga et al., Genes Chrom. Cancer 2001; 30:316-321], pulmonary adenocarcinoma [Sunaga et al., supra], endometrial[Fukuchi et al., Cancer Res. 1998; 58: 3526-3528; Kobayashi et al.,Japan. J. Cancer Res. 1999; 90: 55-59; Mirabelli-Primdahl et al., CancerRes. 1999; 59: 3346-3351], hepatocellular [Satoh et al., supra.; Wong etal., Cancer 2001; 92: 136-145], hepatoblastoma [Koch et al., Cancer Res.1999; 59: 269-273], medulloblastoma [Koch et al., Int. J Cancer 2001;93: 445-449], pancreatic [Abraham et al., Am. J. Pathol. 2002; 160:1361-1369], thyroid [Garcia-Rostan et al., Cancer Res. 1999; 59:1811-1815; Garcia-Rostan et al., Am. J. Pathol. 2001; 158: 987-996],prostate [Chesire et al., Prostate 2000; 45: 323-334; Voeller et al.,Cancer Res. 1998; 58: 2520-2523], melanoma [Reifenberger et al., Int. J.Cancer 2002; 100: 549-556], pilomatricoma [Chan et al., Nature Genet.1999; 21: 410-413], Wilms' tumor [Koesters et al., J. Pathol. 2003; 199:68-76], pancreatoblastomas [Abraham et al., Am. J. Pathol. 2001; 159:1619-1627], liposarcomas [Sakamoto et al., Arch. Pathol. Lab Med. 2002;126: 1071-1078], juvenile nasopharyngeal angiofibromas [Abraham et al.,Am. J. Pathol. 2001; 158: 1073-1078], desmoid [Tejpar et al., Oncogene1999; 18: 6615-6620; Miyoshi et al., Oncol. Res. 1998; 10: 591-594],synovial sarcoma [Saito et al., J. Pathol. 2000; 192: 342-350]. Whileloss-of-function mutations have been associated with cancers such ascolorectal [Fearon et al., Cell 1990; 61: 759-767; Rowan et al., Proc.Natl Acad. Sci. USA 2000; 97: 3352-3357], melanoma [Reifenberger et al.,Int. J. Cancer 2002; 100: 549-556; Rubinfeld et al., Science 1997; 275:1790-1792], medulloblastoma [Koch et al., Int. J. Cancer 2001; 93:445-449; Huang et al., Am. J. Pathol. 2000; 156: 433-437] and desmoids[Tejpar et al., Oncogene 1999; 18: 6615-6620; Alman et al., Am J.Pathol. 1997; 151: 329-334]. Cancers that result from aberrant activityof β-catenin thereby activating the Wnt pathway are suitable fortreatment with the Wnt antagonists of the present invention.

3. Wnt Signaling and Carcinogenesis

The Wnt pathway has many transcriptional endpoints or target genes. Themajority of these are specific to certain types—which is not unusual indevelopmental signaling pathways. This is consistent with a fundamentalmechanism of gene control by extracellular signals in which the cellrather than the signal determines the nature of the response. However,in addition to cell type specific genes, Wnt signaling also controlsgenes that are more widely induced, including components of the Wntsignaling pathway and genes that are most likely activated by theWnt-β-catenin-TCF cascade.

The transition of normal cellular physiology into one characterized byneoplastic change has been the object of intense study in an effort tobetter understand the events underlying the development of cancer. Theinappropriate activation of the target genes by β-catenin thus canresult in a disease state in the organism even though there may not beany somatic mutation in the target genes themselves. Ilyas has generateda modification of the Hanahan and Weinberg list of phenotypes that areacquired by most malignancies; including “Inappropriate stem cellphenotype/limitless replicative potential”, “evasion of apoptosis,”“tissue invasion and metastasis,” “self sufficiency of growth signals,”“insensitivity to growth inhibitors,” “failure of terminaldifferentiation,” “evasion of immune response,” and “sustainedangiogenesis.” Ilyas, J. Pathol. 2005; 205: 130-144; Hanahan andWeinberg, Cell 2000; 100: 57-70. Analysis of the genes modulated by Wntsignaling, including target genes of β-catenin or altered expression asshown by microarray analysis shows that the perturbations from aberrantWnt signaling either directly or through the effect on target genes canimpart nearly all of these “neoplastic phenotypes.” Ilyas, M., J.Pathol. 2005; 205: 130-144. A list of example targets of Wnt signalingis given in Table 1. Gene targets that are upregulated appear inboldface, while those which are downregulated are italicized. Aberrantexpression of such target genes due to the result of activated and/orexcessive Wnt signaling may be remedies upon application of the Wntantagonists of the invention.

Increasingly, cancer is being viewed as a “stem cell” disease (Taipaleet al., Nature 2001; 411: 349-54—that is, an inappropriate activationand/or maintenance of stem cells. Wnt signaling has been shown to beessential for the maintenance of stem cells [He et al., Nature Genet.2004; 36: 1117-1121; Reya et al., Nature 2003; 423: 409-414; Willert etal., Nature 2003; 423: 448-452]. In the intestine, TCF4 is the mainnuclear binding factor for β-catenin and the failure of TCF4 knock outmice to develop stem cells in the small intestine further supports therole of canonical Wnt signaling in stem cell maintenance [Korinek etal., Nat. Genet. 1998; 19: 379-83; Pinto et al., Genes Dev. 2003; 17:1709-13; Kuhnert et al., Proc. Natl. Acad. Sc. USA 2004; 101: 66-71].

The effect of Wnt signaling on multiple biological processes isillustrated by the matrix metalloproteinase genes (MMPs). MMP7, MMP14and MMP26 have been shown to direct targets of β-catenin [Marchenko etal., Int. J. Biochem. Cell Biol. 2004; 36: 942-956; Takahashi et al.,Oncogene 2002; 21: 5861-5867; Brabletz et al., Am. J. Pathol. 1999; 155:1033-1038], while other MMPs were found expressed directly by intestinaladenomas [Paoni et al., Physiol. Genomics 2003; 15: 228-235]. The MMPsare proteolytic enzymes that breakdown stromal collagen thereby allowingtumor cells to acquire the phenotype “tissue invasion and metastasis.”The enzymatic activity also allows the release of latent growth factorsin the stroma, which together with other growth factors secreted by thetumor cells themselves will contribute to “self sufficiency of growthsignals.” [Coussens et al., Science 2002; 295: 2387-2392; Egeblad etal., Nature Rev. Cancer 2002; 2: 161-174]. MMPs can also act onosteopontin (a secondary Wnt-induced target [Paoni et al., supra], torelease fragments which together with vascular endothelial growth factor(VEGF), a direct target of β-catenin, contributes to the feature of“sustained angiogenesis.” [Zhang et al., Cancer Res. 2001; 61:6050-6054; Agnihotri et al., J. Bio. Chem. 2001; 276: 28261-28267].

While the Wnt signaling pathway can be activated at levels downstream ofthe ligand receptor interaction, there is strong evidence to suggestinhibition of the extracellular ligand-receptor interaction component iseffective in reducing the tumorigenicity, even though the eventinitiating the Wnt signaling may have occurred downstream. For example,Ilyas reports in a recent review that the inhibition of Wnt signals inseveral colorectal cancer cell lines results in reduced tumorigenicity.[Ilyas, supra.]. Moreover, the transfection of inoperative frizzledreceptor (Frz7 ectodomain) into carcinoma cell line (SK-CO-1) restored anormal β-catenin phenotype. This cell line has active Wnt signaling dueto a homozygous APC^(−/−) mutation. Moreover, such cells also did notdemonstrate tumor formation when transferred in vivo. Vincan et al.,Differentiation 2005; 73: 142-153. This demonstrates that the inhibitionof Wnt signaling at the extracellular level can downregulate Wntsignaling resulting from activation of a downstream intracellular Wntsignaling pathway component. This further suggests that inhibitors suchas the Wnt antagonists of the present invention, which inhibit Wnt-Frzinteractions, have therapeutic benefit for any Wnt-mediated disorder,regardless of the particular manner in which Wnt signaling has beenactivated.

4. Aberrant Wnt Signaling in Colon Cancer:

Defects in the Wnt signaling component APC was originally discovered tobe the key in the hereditary cancer syndrome familial adenomatouspolyposis (FAP). FAP patients who inherit one defective APC alleledevelop large number of colon polyps, or adenomas, in the early years oftheir life. Such polyps develop as clonal outgrowths of epithelial cellsin which the second APC allele is inactivated. The cumulative effect ofthese FAP adenomas inevitably results in the appearance ofadenocarcinomas, evident as a more or less ordered accumulation ofmutations in additional oncogenes or tumor suppressor genes, such asK-Ras, p53 and Smad4. Moreover, the loss of APC also occurs in mostsporadic colorectal cancers. Kinzler et al., Cell 87: 159-170 (1996).The mutational inactivation of APC, by resulting in the stabilizationof, and eventual nuclear transport of β-catenin, and Wnt signaling,thereby transforms epithelial cells. Interestingly, reporter plasmidscontaining concatemerized TCF binding sites such as the pTOPFLASH,normally transcribed only upon Wnt signaling, are inappropriatelytranscribed in APC mutant cancer cells through constitutive activationof β-catenin/TCF-4 transcription complexes. In other examples ofcolorectal cancer in which APC in not mutated, the scaffolding proteinAxin-2 is mutated [Liu et al., Nature Genet. 26: 146-147 (2000) orβ-catenin is mutated so as to remove the N-terminal Ser/Thr destructionmotif [Morin et al., Science 275: 1787-1790 (1997). Thus, colorectalcancer is linked not only to defects in APC, but to the inappropriatepersistence of β-catenin/TCF-4 transcriptional activation. It hasfurther been reported that TCF-4 mutations result in activation of thesame target genes (as shown by microarray analysis) in colorectalcancers, as is observed through defective APC expression in crypt stemand progenitor cells. Van de Wetering et al., Cell 111: 241-250 (2002).Once the Wnt cascade is activated, the APC^(−/−) adenoma cells maintaintheir progenitor status indefinitely. As a result, it is likely that theactivation of Wnt signaling is a necessary precursor in thecarcinogenesis of colorectal cancer, and the inhibition of Wnt signalingcould be an effective means to treat and/or prevent the onset of thisdisorder.

5. Wnt Signaling in Hematopoietic Stem Cells

Hematopoietic stem cells give rise to the adult blood cells of thecirculatory system in a process of lineage-committed progenitor cellsfrom multipotential hematopoietic stem cells (HSC). It is also apparentthat Wnt signaling contributes to the self-renewal and maintenance ofHSC's, and that dysfunctional Wnt signaling is responsible for variousdisorders resulting from HSC's, such as leukemias and various otherblood related cancers. Reya et al., Nature 434: 843-850 (2005); Baba etal., Immunity 23: 599-609 (2005); Jamieson et al., N Engl. J. Med.351(7): 657-667 (2004). Wnt signaling is normally reduced as stem cellsconvert to committed myeloid progenitor cells. Reya et al., Nature 423:409-414 (2003).

Not only are Wnt ligands themselves produced by HSC's, but Wnt signalingis also active, thereby suggesting autocrine or paracrine regulation.Rattis et al., Curr. Opin. Hematol. 11: 88-94 (2004); Reya et al.,Nature 423: 409-414 (2003). Additionally, both β-catenin and Wnt3apromote self renewal of murine HSCs and progenitor cells, whileapplication of Wnt-5A to human hematopoietic progenitors promotes theexpansion of undifferentiated progenitors in vitro. Reya et al., supra.;Willert et al., Nature 423: 448-452 (2003); Van Den Berg et al., Blood92: 3189-3202 (1998).

In addition to HSC's, it is apparent that embryonic stem cells,epidermal stem cells and epithelial stem cells are responsive ordependent on Wnt signaling for maintenance in an undifferentiated,proliferating state. Willert et al., supra; Korinek et al., Nat. Genet.19: 379-383 (1998); Sato et al., Nat. Med. 10: 55-63 (2004); Gat et al.,Cell 95: 605-614 (1998); Zhu et al., Development 126: 2285-2298 (1999).Therefore the inhibition of Wnt signaling with the Wnt antagonists ofthe present invention may be a therapeutic in the treatment of disordersresulting from dysfunctional hematopoieses, such as leukemias andvarious blood related cancers, such as acute, chronic, lymphoid andmyelogenous leukemias, myelodysplastic syndrome and myeloproliferativedisorders. These include myeloma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's) chronic and nonprogressive anemia, progressive andsymptomatic blood cell deficiencies, polycythemia vera, essential orprimary thrombocythemia, idiopathic myelofibrosis, chronicmyelomonocytic leukemia (CMML), mantle cell lymphoma, cutaneous T-celllymphoma, Waldenstrom macroglobinemia,

6. Wnt Signaling in Leukemia

Unregulated activation of the Wnt signaling pathway is a precursor tothe development of leukemia. Reya et al., supra. Experimental evidenceexists supporting the oncogenic growth of both myeloid and lymphoidlineages as dependent on Wnt signaling. Wnt signaling has beenimplicated in regulating both the chronic and acute forms of myeloidleukemia. Granulocyte-macrophage progenitors (GMPs) from chronicmyelogenous leukemia patients and blast crisis cells from patientsresistant to therapy display activated Wnt signaling. Jamieson, et al.,supra. Moreover, inhibition of β-catenin through ectopic expression ofAxin decreases the replating capacity of leukemic cells in vitro,suggesting that chronic myelogenous leukemia precursors are dependent onWnt signaling for growth and renewal. Also, Wnt overexpression causedGMPs to acquire stem-cell-like properties of long-term self renewal.Jamieson et al., supra. This finding further support the hypothesis thatWnt signaling is necessary for the normal development of blood lineages,but that aberrant Wnt signaling results in the transformation ofprogenitor cells. The Wnt antagonists of the present invention would beuseful to treat these types of leukemias.

Recent studies also suggest that lymphoid neoplasias may also beinfluenced by Wnt signaling. Wnt-16 is overexpressed in pre-B-cellleukemia cell lines carrying the E2A-PbX translocation, suggesting thatautocrine Wnt activity may contribute to oncogenesis. McWhirter, et al.,Proc. Natl. Acad. Sci. USA 96: 11464-11469 (1999). The role of Wntsignaling in the growth and survival of normal B-cell progenitorsfurther supports this notion. Reya et al., Immunity 13: 15-24 (2000);Ranheim et al., Blood 105: 2487-2494 (2005). Autocrine dependence on Wnthas also been proposed for regulating the growth of multiple myeloma, acancer of terminally differentiated B-cells. Derksen et al., Proc. Natl.Acad. Sci. USA 101: 6122-6127 (2004). Primary myelomas and myeloma celllines were also found to express stabilized (i.e., independent ofdegradation complex). Although no mutations in Wnt signaling componentswas present, the overexpression of several components, including Wnt-5Aand Wnt-10B suggest that tumor dependency and cancer self-renewal is notnecessarily dependent on mutations appearing in Wnt signaling pathwaycomponents, but rather only upon constitutive activation of the pathwayitself. Reya et al., supra. Through binding overexpressed Wnt, the Wntantagonists of the present invention would be an effective therapeuticin treating B-cell leukemias.

The transition of self-renewing, pluripotent stem cells to myeloidprogenitors is accompanied by the downregulation of Wnt signaling. Reyaet al, Nature 423: 409-414 (2003). Similarly, the stable expression ofβ-catenin in lymphoid progenitors restored multiple differentiationoptions, albeit such cells lacked markers typically associated witheither cell type. Baba et al., Immunity 23: 599-609 (2005). Thus, it isstrongly suggested that the inhibition of Wnt signaling by the Wntantagonists of the invention could be an effective therapeutic intreating leukemia, such as myelolid and lymphoid leukemia, includingacute and chronic myelogenous leukemia as well as acute and chroniclymphoid leukemias.

7. Aberrant Wnt Signaling in Neural Disorders

It has also been observed that the activation of Wnt signaling throughβ-catenin can increase cycling and expansion of neural progenitors, andthat loss of such signaling can result in a loss of progenitorcompartment. Chenn et al., Science 297: 365-369 (2002); Zechner et al.,Dev. Biol. 258: 406-418 (2003). Just as normal activation of Wntsignaling may promote self-renewal of neuronal stem cells, aberrant Wntpathway activation may be tumorigenic in the nervous system.Experimental evidence supporting this conclusion is the discovery thatmedulloblastoma, a pediatric brain tumor of the cerebellum, containsmutations in both β-catenin and Axin—thereby suggesting thatmedulloblastomas arise from primitive progenitors that becometransformed in response to uncontrolled Wnt signaling. Zurawel et al.,Cancer Res. 58: 896-899 (1998); Dahmen et al., Cancer Res. 61: 7039-7043(2001); Baeza et al., Oncogene 22: 632-636 (2003). Thus, it is stronglysuggested that the inhibition of Wnt signaling by the Wnt antagonists ofthe invention may be an effective therapeutic in the treatment ofvarious neuronal proliferative disorders, including brain tumors, suchas gliomas, astrocytomas, meningiomas, Schwannomas, pituitary tumors,primitive neuroectodermal tumors (PNET), medulloblastomas,craniopharyngioma, pineal region tumors, and non cancerousneurofibromatoses.

8. Aberrant Wnt Signaling in Breast Cancer.

In mammary tissues where stem cells have yet to be definitivelyisolated, a controlling role for Wnt in progenitor cell fate ormaintenance is suggested by studies of Wnt transgenic mice developmammary tumors. These tumors have an increased frequency of individualcells with stem and progenitor properties, in stark contrast to tumorsfrom mice overexpressing other oncogenes. [Liu et al., Proc. Natl. Acad.Sci. USA 101: 4158-4163 (2004); Li et al., Proc. Natl. Acad. Sci. USA100: 15853-15858 (2003)]. This suggests that the Wnt pathway may beunique in its ability to target stem and progenitor cells fortransformation, and suggests a key role in the self-renewal of normalbreast epithelium. Thus the inhibition of Wnt signaling by the Wntantagonists of the invention is likely an effective therapeutic in thetreatment of breast cancer.

FIG. 32 is an illustration of active Wnt signaling in human breastcancer. FIG. 32A shows Wnt-1 expression (as shown by in vitrohybridization) in normal (A-1), low grade (A-2) and high grade (A-3)human breast tumor initially reported in Wong et al., J. Pathol. 196:145 (2002). FIG. 32B shows nuclear (B-1) and cytoplasmic (B-2)localization (as shown by IHC) of β-catenin in breast cancer patients.Also shown is a Kaplan-Meier survival plot (B-3) showing patientsurvival probability that correlates with the indicated β-cateninexpression pattern. This data was initially reported in Lin et al.,P.N.A.S. (USA) 97(8): 4262-66 (2000). FIG. 32C is a microarray analysisof Wnt-1 expression in a normal breast from a patient without cancer incomparison with tissue isolated from a patient with infiltrating ductalcarcinoma, her-2 negative.

9. Wnt Signaling in Aging

The Wnt signaling pathway may also play a critical role in aging andage-related disorders.

As reported in Brack A S, et al., Science, 317(5839):807-10 (2007),muscle stem cells from aged mice were observed to convert from amyogenic to a fibrogenic lineage as they begin to proliferate. Thisconversion is associated with an increase in canonical Wnt signalingpathway activity in aged myogenic progenitors and can be suppressed byWnt inhibitors. Additionally, components of serum from aged mice bind tothe Frizzled proteins and may account for the elevated Wnt signaling inaged cells. Injection of Wnt3A into young regenerating muscle reducedproliferation and increased deposition of connective tissue.

The Wnt signaling pathway has been further implicated in aging processin studies using the Klotho mouse model of accelerated aging in which itwas determined that the Klotho protein physically interacted with andinhibited Wnt proteins. Liu H, et al., Science, 317(5839):803-6(2007).In a cell culture model, the Wnt-Klotho interaction resulted in thesuppression of Wnt biological activity while tissues and organs fromKlotho-deficient animals showed evidence of increased Wnt signaling.

Accordingly, Wnt antagonists could find use as therapeutics to reducethe effects of aging and to treat age-related diseases.

I. Modes of Administration Specific Formulations 1. GeneralConsiderations

A pharmaceutical composition is formulated to be compatible with itsintended route of administration, including intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid (EDTA); bufferssuch as acetates, citrates or phosphates, and agents for the adjustmentof tonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampules, disposable syringesor multiple dose vials made of glass or plastic.

2. Injectable Formulations

Pharmaceutical compositions suitable for injection include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOREL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid so as to beadministered using a syringe. Such compositions should be stable duringmanufacture and storage and must be preserved against contamination frommicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(such as glycerol, propylene glycol, and liquid polyethylene glycol),and suitable mixtures. Proper fluidity can be maintained, for example,by using a coating such as lecithin, by maintaining the requiredparticle size in the case of dispersion and by using surfactants.Various antibacterial and antifungal agents; for example, parabens,chlorobutanol, phenol, ascorbic acid, and thimerosal, can containmicroorganism contamination. Isotonic agents; for example, sugars,polyalcohols such as manitol, sorbitol, and sodium chloride can beincluded in the composition. Compositions that can delay absorptioninclude agents such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., any modulator substance/molecule of the invention) inthe required amount in an appropriate solvent with one or a combinationof ingredients as required, followed by sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle that contains a basic dispersion medium, and the otherrequired ingredients. Sterile powders for the preparation of sterileinjectable solutions, methods of preparation include vacuum drying andfreeze-drying that yield a powder containing the active ingredient andany desired ingredient from a sterile solutions.

3. Systemic Administration

Systemic administration can also be transmucosal or transdermal. Fortransmucosal or transdermal administration, penetrants that can permeatethe target barrier(s) are selected. Transmucosal penetrants include,detergents, bile salts, and fusidic acid derivatives. Nasal sprays orsuppositories can be used for transmucosal administration. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams.

The compounds can also be prepared in the form of suppositories (e.g.,with bases such as cocoa butter and other glycerides) or retentionenemas for rectal delivery.

4. Carriers

In one embodiment, the active compounds are prepared with carriers thatprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable or biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Such materials can beobtained commercially from ALZA Corporation (Mountain View, Calif.) andNOVA Pharmaceuticals, Inc. (Lake Elsinore, Calif.), or prepared by oneof skill in the art. Liposomal suspensions can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, such as in (Eppstein et al.,U.S. Pat. No. 4,522,811, 1985).

5. Unit Dosage

Oral formulations or parenteral compositions in unit dosage form can becreated to facilitate administration and dosage uniformity. Unit dosageform refers to physically discrete units suited as single dosages forthe subject to be treated, containing a therapeutically effectivequantity of active compound in association with the requiredpharmaceutical carrier. The specification for the unit dosage forms aredictated by, and directly dependent on, the unique characteristics ofthe active compound and the particular desired therapeutic effect, andthe inherent limitations of compounding the active compound.

6. Gene Therapy Compositions

The nucleic acid molecules can be inserted into vectors and used as genetherapy vectors. Gene therapy vectors can be delivered to a subject by,for example, intravenous injection, local administration (Nabel andNabel, U.S. Pat. No. 5,328,470, 1994), or by stereotactic injection(Chen et al., Proc Natl Acad Sci USA. 91:3054-7 (1994)). Thepharmaceutical preparation of a gene therapy vector can include anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells that produce the gene delivery system.

7. Dosage

The pharmaceutical composition and method may further comprise othertherapeutically active compounds that are usually applied in theadministration of the Wnt antagonists.

In the treatment or prevention of conditions which requireadministration of Wnt antagonists, an appropriate dosage level willgenerally be about 0.01 to 500 mg per kg patient body weight per daywhich can be administered in single or multiple doses. Preferably, thedosage level will be about 0.1 to about 250 mg/kg per day; morepreferably about 0.5 to about 100 mg/kg per day. A suitable dosage levelmay be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day,or about 0.1 to 50 mg/kg per day. Within this range the dosage may be0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day. For oral administration,the compositions are preferably provided in the form of tabletscontaining 1.0 to 1000 milligrams of the active ingredient, particularly1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0,250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0milligrams of the active ingredient for the symptomatic adjustment ofthe dosage to the patient to be treated. The compounds may beadministered on a regimen of 1 to 4 times per day, preferably once ortwice per day.

However, the specific dose level and frequency of dosage for anyparticular patient may be varied and will depend upon a variety offactors including the activity of the specific compound employed, themetabolic stability and length of action of that compound, the age, bodyweight, general health, sex, diet, mode and time of administration, rateof excretion, drug combination, the severity of the particularcondition, and the host undergoing therapy.

8. Kits for Compositions

The compositions (e.g., pharmaceutical compositions) can be included ina kit, container, pack, or dispenser together with instructions foradministration. When supplied as a kit, the different components of thecomposition may be packaged in separate containers and admixedimmediately before use. Such packaging of the components separately maypermit long-term storage without losing the active components'functions.

Kits may also include reagents in separate containers that facilitatethe execution of a specific test, such as diagnostic tests or tissuetyping.

(a) Containers or Vessels

The reagents included in kits can be supplied in containers of any sortsuch that the life of the different components are preserved and are notadsorbed or altered by the materials of the container.

For example, sealed glass ampules may contain lyophilized modulatorsubstance/molecule and/or buffer that have been packaged under aneutral, non-reacting gas, such as nitrogen. Ampules may consist of anysuitable material, such as glass, organic polymers, such aspolycarbonate, polystyrene, etc., ceramic, metal or any other materialtypically employed to hold reagents. Other examples of suitablecontainers include simple bottles that may be fabricated from similarsubstances as ampules, and envelopes, that may consist of foil-linedinteriors, such as aluminum or an alloy. Other containers include testtubes, vials, flasks, bottles, syringes, or the like. Containers mayhave a sterile access port, such as a bottle having a stopper that canbe pierced by a hypodermic injection needle. Other containers may havetwo compartments that are separated by a readily removable membrane thatupon removal permits the components to mix. Removable membranes may beglass, plastic, rubber, etc.

(b) Instructional Materials

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, laserdisc, audio tape, etc. Detailed instructions maynot be physically associated with the kit; instead, a user may bedirected to an internet web site specified by the manufacturer ordistributor of the kit, or supplied as electronic mail.

9. Combination Therapy

In certain embodiments, a pharmaceutical formulation comprising a Wntantagonist is administered in combination with at least one additionaltherapeutic agent and/or adjuvant. In certain embodiments, theadditional therapeutic agent is a chemotherapeutic agent, growthinhibitory agent, or cytotoxic agent like a toxin, such as amaytansinoid, calicheamicin, antibiotic, radioactive isotope,nucleolytic enzyme or the like.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of a Wnt antagonist can occur prior to, simultaneously,and/or following, administration of the additional therapeutic agentand/or adjuvant. A Wnt antagonist can also be used in combination withradiation therapy.

10. Medicaments

The invention provides a Wnt antagonist for a use in the preparation ofa medicament useful for treating a Wnt-mediated disorder. In a specificaspect, the Wnt-mediated disorder is cancer.

The following examples are included to demonstrate preferred embodimentsof the present invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments that are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention. All references cited throughout thespecification are expressly incorporated by reference in their entiretyherein.

Example 1 General Protocols Mammalian Cell Culture.

Human kidney epithelial (HEK) 293 cells (ATCC # CRL-1573), human ovarianPA1 cells (ATCC # CRL-1572) were grown in 50/50 Dulbecco modified Eaglehigh glucose medium, Ham's F12 which has been supplemented with 10%fetal bovine serum. Human teratoma derived NTer2 (ATCC #CRL-1973) andTera2 (ATCC#HTB-106) cells were maintained in McCoy's mediumsupplemented with 15% fetal bovine serum and NCCIT cells (ATCC #CRL-2073) were maintained in RPMI supplemented with 10% fetal bovineserum. All cell lines were further supplemented with 2 mM glutamine, and1% penicillin-streptomycin at 37° C. in 5% CO₂.

Transfection and Luciferase Assays

In preparation for transfection, (1) 500,000 HEK293 and (2) 100,000 PA1cells (ATCC # CRL-1572), NCCIT, NTera2 or Tera2 cells were plated intoeach well of a 12-well dish (Nuc) 24 hours before transfections. Cellswere transfected with 0.375 μg TOPglow (Upstate, Cat #21-204), 0.05 mgLEF1, 0.01 mg SV40 RL with Fugene (Roche) and at 24 hours posttransfection. Media was changed and cells were untreated or treated withWnt3a alone, Wnt-5a alone, or with serum samples for an additional 20-24hours before harvesting. All dilutions were made in complete media forthe indicated cell lines. Cells were harvested in 50-100 μl of 1×SJClysis buffer (20 mM Tris pH 8.0, 137 mM NaCl, 1 mM EGTA, 1% TritonX-100, 10% Glycerol, 1.5 mM MgCl₂, 1 mM DTT, 50 mM NaF, 1 mM NaVO4 andprotease inhibitors) and duplicate 10 μl were assayed using Dual-Glo™luciferase assay kit (Promega, Part # TM058) and detected in an EnvisionLuminometer (Perkin Elmer). Luciferase activity was normalized againstRenilla activity.

Example 2 Construction of Frz-Fc Chimeric Molecules Cloning andExpression Frz8(173)-Fc and Frz8(156)-Fc

FIGS. 4A and B show the sequences of the Frz8 (156)-Fc and Frz8 (173)-Fcchimeric constructs. FIG. 4A shows the longer Frz8(173) sequence. Shownin gray (i.e., first 24 N-terminal amino acid residues) is the leadersignal sequence. Shown in underline (i.e., residues 25-27) are alanineresidues that may be present or absent in the mature protein. Shown inboxed text (i.e., residues 157-173) are the additional sequences of theFrz8 receptors that distinguish the longer Frz8 (173) from the shorterFrz8(156) chimeric constructs. Shown in bold (i.e., residues 174-182) isthe linker sequence, while the sequence in italics (i.e., residues183-409) is the Fc region. FIG. 4B shows the shorter Frz (156) minimalCRD (ECD) domain sequence. In gray (i.e., first 24 N-terminal amino acidresidues) is the leader signal sequence. Shown in underline (i.e.,residues 25-27) are alanine residues that may be present or absent inthe mature protein. Shown in bold (i.e., residues 157-164) is the linkersequence, while the sequence in italics (i.e., residues 165-391) is theFc region.

The Frz8(156)-Fc construct was constructed as follows. cDNA encodingFrizzled 8 residues 1-156 were sub-cloned into the EcoR1 and XhoI sitesof a pRK-derived plasmid. While native human cDNA is preferred,alternative sequence encoding identical protein sequence (e.g., murine)may also be used. In this cloning procedure, the carboxyl terminus ofthe Frz8 was fused to the amino terminus of a human IgG effector domain(Fc) via a short linker region (e.g., residues LESGGGGVT)(SEQ ID NO:70), to create an Frz8-Fc fusion. A final construct encodes 156 residuesof Frz8. The cloning was performed using standard molecular biologytechniques (Ausubel et al. (eds.), 2003, Current Protocols in MolecularBiology, 4 Vols., John Wiley & Sons). Protein was expressed in ChineseHamster Ovary (CHO) cells.

Alternatively, the cDNA encoding a length of Frz8 of a length differentthan described previously (e.g., 1-173) may be used. In addition, analternative linker sequence (e.g., ESGGGGVT)(SEQ ID NO: 69) may also beused.

Frz-Fc and sFrp-Fc Constructs

The constructs for the Wnt antagonists with a Frizzled domain componentcomprising Frz1, Frz2, Frz3, Frz4, Frz5, Frz6, Frz7, Frz9, Frz10, sFRP1,sFRP2, sFRP3, sFRP4, or sFRP5 were constructed in a manner similar tothe procedure described for Frz8. Frz2, Frz3, Frz4, Frz5, and sFRP3 weresubcloned into a pRK-derived plasmid using XhoI and AscI. Frz1, Frz6,Frz7, Frz9, Frz10, and sFRP4 were subcloned into a pRK-derived plasmidusing ClaI and XhoI and sFrp1, sFrp2, and sFRP5 were subcloned into apRK-derived plasmid using ClaI and AscI. As with the Frz8 constructs,the carboxyl terminus of the Frz domains were fused to the aminoterminus of a human IgG effector domain (Fc) via a short linker regionto create the chimeric Wnt antagonists. FIGS. 7 (A, B, and C) showsexemplary amino acid sequences for these constructs. The leader signalsequence is shown in bold with italics indicating a non-native leadersequence. The linker is underlined and the Fc component is shown initalics.

FIG. 5 (A-H) (SEQ ID NOs: 115-129) provides exemplary nucleic acidsequences for these Wnt antagonist constructs.

Alternative constructs can be made to optimize in vivo activity orstability or to provide other beneficial characteristics, such as, forexample, increased solubility, improved binding characteristics. Theseconstructs may include linkers that are different than the linkers ofthe above-described Wnt antagonists. For example, an alternativeconstruct of the Frz3-Fc chimeric protein (SEQ ID NO: 114) has been madeby subcloning a Frz3 domain into a pRK-derived plasmid using BstXI andXhoI and using the LESGGGGVT (SEQ ID NO: 70) peptide linker to fuse theFrz3 domain to the Fc domain.

Protein Isolation

The Wnt antagonist chimeric proteins were isolated to >90% purity byaffinity capture using a PROSEP® (Millipore) protein-A conjugated resin.Higher order aggregates were separated from dimers by passage over aSuperdex 200® (GE-Healthcare) gel-filtration column. Protein identityand processing of the amino terminus to remove the signal sequence wereconfirmed by Edmund degradation. Purity of the final protein isestimated to be greater than 98% (FIG. 10). Endotoxin levels of thematerial after purification is complete and less than 1.0 EU/mg.

Example 3 Serum Stability of Frz8-Fc Chimeric Molecules

Initial studies of the serum stability of the Frz8(173)-Fc chimericconstructs indicated that the construct had a limited in vivo half-life.The in vivo instability was likely due to the presence of proteasecleavage sites in the EC domain (ECD) of the Frizzled receptorcomponent. The Frz8(156)-Fc construct described in Example 2 exhibitedincreased serum stability over the Frz8(173)-Fc. Athymic nude mice wereinjected i.v. with 10 mg/kg of either Frz8(173)-Fc or Frz8(156)-Fc.Serum was collected at specified time points and analyzed for total andactive protein. FIG. 11A shows an immunoblot for human Fc used to detectthe protein present in 1 μL of serum and compared with 25 μg of therespective purified protein (P). Frz8(156)-Fc was detectable in serum 72h after administration, whereas Frz8(173)-Fc was not detectable beyond30 minutes.

The activity of Frz8(156)-Fc and Frz8(173)-Fc in the collected serum wasassayed by measuring the inhibition of Wnt3a-dependent TOPglow reporteractivity in HEK293 cells. Although comparable in vitro potency wasobserved on treatment with purified Frz8(156)-Fc and Frz8(173)-Fc at 2.5μg/mL, only partial inhibitory activity was recovered from the serum ofFrz8(173)-Fc-treated mice collected 30 minutes after proteinadministration. In contrast, more potent inhibitory activity could berecovered from the serum of Frz8(156)-Fc treated mice for up to 24 hoursafter administration, with detectable levels of inhibition for at least72 hours (FIG. 11B). These studies demonstrate that the Frz8(156)molecule is more stable in vivo than the molecule based on Frz8(173).

Additionally, the Frz8(173)-FC had suboptimal efficacy and acted only toreduce the rate of increase in tumor volume, as opposed to shrinkingstarting tumor volume. This suboptimal efficacy is illustrated in FIG.12, showing a graph of tumor volume over time resulting from treatmentwith various Wnt signaling component-Fc chimeric antagonists, includingthe Frz8(173)-FC molecule. In this assay, the MMTV-WNT-1 tumors weretransplanted into the mammary fat pad of athymic nude mice, and drug wasadministered IV at the time points indicated by the arrows on theX-axis.

Example 4 In Vivo Pharmacokinetics of Frz8(156)-FC

The in vivo pharmacokinetics of Frz8(156)-FC were tested byadministration of a single dose of this protein at 1, 5, or 20 mg/kgi.v. or at 20 mg/kg i.p. into nude mice. As reported in FIG. 13 anddiscussed further in this Example below, the Frz8-Fc reagent displayedbiphasic elimination in nude mice at all doses. After a single IV or IPdose, Frz8-Fc displays: (1) dose proportional increase in exposure; (2)rapid absorption after IP dosing; (3) clearance of about 25-30 ml/day;and (4) a half life of about 4 days. Bioavailability coefficient,AUC_(IP)/AUC_(IV)=92%.

Animal Protocol

Female athymic nude mice are separated into 4 groups of 12, on the basisof quantity of drug administered and manner of administration. Group 1:Frz8-Fc 1 mg/kg, intravenous (IV); Group 2: Frz8-Fc, 5 mg/kg, IV; Group3: Frz8-Fc 20 mg/kg, IV; and Group 4: Frz8-Fc, 20 mg/kg, interperitoneal(IP). Each animal received an IV or IP bolus dose of Frz8-Fc accordingto the group designation. The dose volume administered (5-10 mL/kg)varies depending upon the concentration of the dosing solution and theweight of each animal. IV dosing is administered via the tail vein.

About 125 μl of blood is collected from each animal according to thefollowing procedure. Serum is stored at −70° C. until assayed by ELISA.Sample are drawn such that n=3 animals/timepoint. Extra animals are usedfor predose sample collection and/or collection of blank mouse serum.Blood is collected with a retroorbital bleed for the first twotimepoints for each animal, using alternative eyes. For the finaltimepoint, blood is collected via a cardiac stick and about 1 ml isaliquoted into 2 tubes. One sample will be used to determine Frz8-Fcconcentration and the other will be reserved for research use. Eachanimal receives an IP bolus of 10 ml saline as fluid replacement aftereach blood collection timepoint. Retroorbital bleeds are performed underisoflorane anesthesia and terminal bleeds occur under aketamine/xylazine cocktail. Animals are euthanized via cervicaldislocation under anesthesia after the final blood draw.

Results

FIG. 13A is an immunoblot of a neat serum from mice treated with Frz8-Fcshowing detection in serum at 7 days and beyond from both 20 or 5 mg/kgI.V. or 20 mg/kg I.P. Samples were taken from individual mice at 4, 7,10 or 14 days. For controls, serum samples were taken from untreatedmice, Frz8-Fc protein was added to 20 μg/ml and the samples incubatedfor 2 hours at 37° C. and the sample was then treated with SDS loadingbuffer (labeled as 2 h); neat serum from untreated mice was also run asa negative control (labeled as S).

FIGS. 13B and 13C are a graphical summary of Frz8-Fc serum levels asdetermined from the pharmacokinetic study. Specific periods of timeinclude evaluation over 16 days (FIG. 13B) and 2 days (FIG. 13C).Frz8-Fc displayed biphasic elimination administration in nude mice atall doses. Curves represent the predicted concentrations, whileindividual data points represent the average serum levels of Frz8-Fcprotein from individual mice as determined by ELISA. FIG. 13D is asummary of the parameters for a biphasic model of Frz8-Fcpharmacokinetics. When dosed at 20 mg/kg by either the i.p. or i.v.route, comparable serum levels of protein were achieved within a day ofinjection and the protein was detectable in serum up to 7 days. Afteri.p. dosing at 20 mg/kg, protein was rapidly absorbed with a T_(max) of˜8 h and bioavailability (AUC_(IP)/AUC_(IV)) of 92%. The clearance ofthe protein was ˜25 to 30 mL/d/kg with a half-life of about 4 days

Example 5 Binding Affinity of Frz-FC Molecules

The addition of the FC domain to the Frz8(156) domains results in anincrease in binding affinity for Wnt3a of over two magnitudes. FIG. 14demonstrates the enhanced ability of Frz8-ECD to block Wnt3a signalingwhen linked to a dimeric Fc domain. FIG. 14A is an IC₅₀ graph of a Wnt3ainhibition assay of two different preparations of Frz8(156)-FC. FIG. 14Bis a gel confirming the purity of the isolated Frz8(156) CRD (ECD).Shown are: (a) non-reduced Frz8(156) ECD (Lane 1); (b) molecular weightmarkers (Lane 2); and reduced Frz8 ECD (156) (Lane 3). This gelindicates that the Frz8 ECD used in the binding assay is intact and runsat approximately the expected molecular weight.

Example 6 Binding Activity of Frz-FC Chimeras ELISA

For PK evaluation of the Wnt antagonist, the wells of a 384-well ELISAmicro titer plate (Nunc Maxisorp, Rochester, N.Y.) were coated with therabbit anti-human Fc (Jackson Immuno Research, Westgrove, Pa.) at aconcentration of 1 μg/m′ in PBS (25 μg/well). After an overnightincubation at 4° C., the rabbit anti-human Fc solution was decanted, andthe plates were blocked with 40 μl/well of block buffer (PBS containing0.5% BSA and 10 ppm proclin). After a 60 minute incubation at roomtemperature with gentle agitation, the rabbit anti-human Fc coatedplates were washed three times with wash buffer (PBS 0.05% Tween 20® and10 ppm proclin). The Frizzled-Fc standards (a dilution series with aconcentration range of 0.78-100 ng/ml), and the samples diluted intoassay range in assay buffer (PBS containing 0.5% BSA, 0.05% Tween 20®and 10 ppm proclin) were added to the assay plate (25 μl/well). After a120 minute incubation at room temperature with gentle agitation, theassay plates were washed six times with wash buffer. The remaining boundFrz-Fc was detected using a horse radish peroxidase (HRP) conjugatedgoat anti-human IgG-Fc (Jackson Immuno Research) diluted into assaydiluent (25 μl/well). After appropriate color development (10-25minutes) the enzymatic reaction was stopped with 1M phosphoric acid (25μl/well). The assay plates were read at a wavelength of 450 nm with areference wavelength of 630 nm. Sample concentrations were determined bycomparing the sample OD against the standard curve fit using a4-parameter algorithm.

BIAcore

FIG. 15 demonstrates direct binding by Wnt3a to the Frz8(1-156)-Fcchimera. This chimera protein was amine coupled to a Biocore™ (BIAcore,Inc. Piscataway, N.J.) CMS sensor chip at approximately 1700 responseunits as described generally in Chen, Y. et al., J. Mol. Biol. 293:865-881 (1999). Briefly, carboxymethylated dextran biosensor chips (CMS,BIAcore™ Inc.) were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions. Aninjection of 1M ethanolamine was done to block unreacted groups. Wnt3awas then injected at an estimated concentration of 0.5 μg/ml and bindingwas assessed by the change in response units as a function of time.Wnt3a was found to bind Frz8-Fc. As shown in FIG. 15, the association ofWnt3a and Frz-Fc results in a highly significant increase of 1000response units over control protein (E. coli expressed non-nativeWnt3a.)

OCTET

The ability of the Wnt antagonists to interact with the Wnt ligandsWnt3a and Wnt5a was measured using the OCTET™-QK system. (FortéBio,Inc., Menlo Park, Calif.). This system allows for the measurement ofprotein binding at a biosensor surface. The assays were conducted byfirst incubating one of the Wnt antagonist molecules (20 ug/mL) withanti-human IgG Fc-specific biosensors for 10 minutes in phosphatebuffered saline (PBS) with 0.5% CHAPS. The unbound Wnt antagonist wasremoved by washing for 1.5 minutes in PBS 0.5% CHAPS. Either Wnt3a orWnt5a (5.0 ug/mL) was then added to the assay and incubated with the Wntantagonist molecules bound to the biosensor surface for 5 minutes in PBSwith 0.5% CHAPS. The interaction between the Wnt antagonist moleculesand Wnt ligand was monitored in the same buffer. All assay steps wereperformed at room temperature in a volume of 150 uL. FIG. 16 shows theresult of this binding assay with FIG. 16A showing data from the bindingof Wnt3a to the Frz1-Frz10-Fc chimeras, FIG. 16B showing data from thebinding of Wnt3a to sFRP-Fc chimeras, and FIG. 16C showing data from thebinding of Wnt5a to the Frz1-Frz10-Fc chimeras and sFRP-Fc chimeras.

The OCTET™ assay indicates that both Wnt3a and Wnt-5a bind Fz8-Fc,Fz5-Fc, and Fz4-Fc the fastest, relative to the other Frz proteins, withWnt3a binding Fz1-Fc, Fz2-Fc, and Fz7-Fc at a slower rate. The amplitudeand linear nature of Wnt-5a binding curves suggest a lower bindingaffinity relative to Wnt3a binding, as determined by this binding assay.The amplitude of the OCTET™ binding data suggest that the sFRP-Fcproteins have an affinity for Wnt3a similar that observed for Frz1,Frz2, and Frz7, and somewhat lower that observed for Frz5 and Frz8.

Example 7 Inhibition of Wnt Signaling by the Wnt Antagonists—CellularAssays

Cellular assays were performed using 293 (human kidney) cellstransfected with the TOPglow reporter plasmid. In preparation fortransfection approximately 500,000 HEK293 were plated into a well of a12-well dish (Nuc) 24 hours before transfections. Cells were transfectedwith 0.375 μg TOPglow (Upstate, Cat #21-204), 0.05 mg LEF1, 0.01 mg SV40RL with Fugene (Roche) and at 24 hours post transfection. Media waschanged and cells were untreated or treated with Wnt3a alone, Wnt-5aalone, or with a Wnt antagonist for an additional 20-24 hours beforeharvesting. All dilutions were made in complete media for the indicatedcell lines. Cells were harvested in 50-100 μl of 1×SJC lysis buffer (20mM Tris pH 8.0, 137 mM NaCl, 1 mM EGTA, 1% Triton X-100, 10% Glycerol,1.5 mM MgCl₂, 1 mM DTT, 50 mM NaF, 1 mM NaVO4 and protease inhibitors)and duplicate 10 μl were assayed using Dual-Glo™ luciferase assay kit(Promega, Part # TM058) and detected in an Envision Luminometer (PerkinElmer). Luciferase activity was normalized against Renilla activity.

Cells to be treated with Wnt5a were transfected with Frz4 and Lrp5 inaddition to the reporter. The presence of these additional componentsallows Wnt pathway activation by Wnt5a to proceed as per the canonicalpathway. Mikels A J, and Nusse R., PLOS Biol. 4:e115 (2006). Wnt3aactivated cells were treated with 100 ng/ml Wnt3a and Wnt5a activatedcells were treated with 1 ug/ml Wnt5a.

As shown in FIGS. 17A and B, the Frz-Fc antagonist inhibited Wntsignaling to varying degrees. Both Frz5-Fc and Frz8-Fc showed completeinhibition of the Wnt3a signal and significantly inhibited the Wnt5asignal. Frz4-Fc, Frz2-Fc, and Frz7-Fc showed significant inhibition ofthe Wnt3a signal.

Example 8 Relative IC50s of the Wnt Antagonists

The relative IC50s of the Wnt antagonists were determined by measuringinhibition of Wnt signaling by the Wnt antagonists in U2OS (humanosteosarcoma) cells stably transfected with TOPglow luciferase TCFreporter plasmid as described in Example 7. Initial Wnt signaling incells was obtained with Wnt3a activation. A 3-fold dilution series ofFrz-Fcs was applied to cells overnight. (FIG. 18). As determined by thisassay, Wnt3a binds to Frz8-Fc, Frz5-Fc, and Frz4-Fc with sub-nanomolarIC50 (with Frz8-Fc having an IC50 of 0.04 nM, Frz-5Fc having an IC50 of0.20 nM, and Frz-4 having an IC50 of 0.48 nM) and to Frz2-Fc and Frz7-Fcwith nanomolar IC50 (with Frz2-Fc having an IC50 of 1.2 nM and Frz2-Fchaving an IC50 of 1.4 nM.

Example 9 Wnt Target Genes as Pharmacodynamic Markers of Drug Response

As an alternative to immunohistochemical analysis of β-catenin,transcriptional targets of Wnt were used to monitor inhibition of Wntsignaling activity. The cell lines that had autocrine Wnt signalingshowed increased expression of known Wnt target genes and thisexpression was regulated by in vitro treatment with Wnt3a as well as byFrz8-Fc. RNA analysis of NTera-2 cells indicated that Frz8-Fc treatmentaffects expression of the Wnt target genes tested. Thus, the expressionof these genes can be followed as an indicator of treatment efficacy. Asan extension of these observations, expression of these Wnt target genescan be used as a diagnostic tool to identify cancers that are driven byWnt signaling and are likely candidates for anti-Wnt therapeutic agents.

In Vitro

In vitro comparative gene expression analysis on PA-1 cells treated withpurified Wnt3a, Fz8 CRD-Fc, or a control protein was performed todetermine the suitability of Wnt target genes to indicate in vivoinhibition of Wnt signaling in teratoma cells. RNA isolated from PA-1cells that were treated with Wnt3a, Frz8-Fc, or control Fc protein wassubject to microarray analysis and the change in expression levels ofthe indicated genes in response to exogenously added Wnt3a, Frz8-Fc, andcontrol Fc protein was determined. For microarray analysis, cells weretreated with the indicated proteins in triplicate and total RNA wasisolated using the RNAeasy kit (Qiagen). Array analysis was done on theAffymetrix Human Genome U133 Gene Chip set (Rubinfeld B, et al., NatBiotechnol 2006; 24:205-9). The specific probes and primer sets areshown in FIG. 20.

The expression levels of previously identified targets of Wnt signalingsuch as Axin2, APCDD1, and Gad1 were up-regulated by Wnt3a treatment ordown-regulated by Frz8-Fc treatment (FIG. 19A). Moreover, some genessuch as Lefty2 (A), Lefty′ (B), sFRP1, and Fzd5 were down-regulated byWnt3a and up-regulated by inhibition of Wnt signaling with Frz8-Fc (FIG.19A). Subsequent gene expression analysis by qRT-PCR showed that thesetranscripts were similarly regulated by Wnt3a and Fz8 CRD-Fc in NTera-2,Tera-2, and NCCIT cells as well.

In Vivo

APCDD1, Gad1, and Fzd5 were among the most consistently modulated genesin above described in vitro analyses and were therefore selected aspotential markers of Wnt responsiveness for in vivo tumor xenograftstudies.

Tumor tissue RNA was purified from xenograft specimens collected at theend of the efficacy study and quantitative reverse transcription-PCR(qRT-PCR) analysis of Wnt-responsive transcripts carried out aspreviously described Rubinfeld B, et al., Nat Biotechnol 2006;24:205-9). Fold induction for each gene was determined using the ΔΔCtmethod and the result presented relative to glyceraldehyde-3-phosphatedehydrogenase. The specific probes and primer sets are shown in FIG. 20.All reactions were done in duplicate and the average of at least twoassays±SEM was plotted.

Similar to the effects seen in vitro, treatment with therapeutic dosesof Frz8-Fc reduced the expression of genes for APCDD1 and Gad1 andincreased the expression of Fzd5 in tumors from the NTera-2 xenografts(FIG. 19B). Although there is a general nonspecific down-regulation ofall genes following CD4hFc treatment, these changes were notstatistically significant compared with those seen in Frz8-Fc-treatedtumors. These observations show that the antitumorigenic effects ofFrz8-Fc in vivo are on target genes and that the expression levels ofthese genes can be used to monitor the efficacy of potential anti-Wnttherapeutic agents.

Example 10 Inhibitory Effect of Wnt Antagonists on Growth of Tumors inMice with Allografts and Human Xenografts

The studies set forth in this Example indicate that the Wnt antagonistsare useful in treating Wnt expressing tumors. The largely complete tumorregression in the case of Wnt-1-MMTV model illustrate the effect of theWnt antagonists on tumors that are strongly Wnt driven. However, thesignificant effect of the Wnt antagonist on the PA-1 and NTer2 tumorsalso reflects the strong therapeutic potential to treat tumors that maynot be entirely Wnt driven.

Animals

Female C57B16 mice (The Jackson Laboratory) were used for the passagingof MMTV-Wnt1 tumors. Maintenance of mice and in vivo procedures werecarried out using Institutional Animal Care and Use Committee-approvedprotocols.

MMTV-Wnt Model-Allografts

FIG. 21 is a linear schematic describing the vector construct used inthe transfection to create the Wnt animal model. This construct mimicsthe constitutive Wnt signaling activation observed with MMTA viralinsertion, as described in Tsujomoto et al., Cell 55: 619-625 (1988) andLi et al., Oncogene 19: 1002-1009 (2000).

Passaging of MMTV-Wnt1 Transgenic Tumors in Mice.

The tumors from MMTV-Wnt1 transgenic mice were serially passaged inC57B16 mice for 6 to 10 passages by surgical implantation in the mammaryfat pad. Tumor tissue was aseptically collected from the transgenicmouse, rinsed in HBSS and cut into small pieces. The recipient mice wereanaesthetized with a mixture of ketamine (75-80 mg/kg) and xylazine(7.5-15 mg/kg), the tumor fragment inserted under the skin rostral tothe third mammary fat pad, and the skin closed using wound clips. Tumorswere passaged for a maximum of 10 passages, and after the first twopassages, tumor tissue was examined histologically to confirm that itwas of mammary origin and continues to express Wnt. Mammaryadenocarcinomas develop in 6-12 months in the mice. Tumors isolated fromthese mice were used to create the transplant models described below.

In Vivo Studies.

For in vivo studies testing the efficacy of Wnt antagonists in theMMTV-Wnt model, the tumor cells were introduced by subcutaneousinjection of cells obtained from macerated tumors tissue. Tumor tissuewas aseptically collected from mice transplanted with tumors from Wnttransgenic mice (described above), rinsed in PBS or HBSS, cut intosmaller pieces and macerated into HBSS using a cell dissociation kit(Sigma). The cells were washed twice in sterile HBSS and suspended in a50% matrigel solution in HBSS. The cell suspension was inoculatedsubcutaneously into the mammary fat pad of athymic nude mice, with avolume not exceeding 150 μl/mouse.

For in vivo studies using the NTera2 or PA-1 animals models, cells weregrown as described and harvested when growth is in the logarithmicphase. The cells were suspended in a 50% matrigel solution in HBSS andinoculated subsutaneously into athymic nude mice at a concentration ofeither 8 million cells/mouse (NTera2) or 10 million cells/mouse (PA-1).

Tumors were monitored daily and measured after 7-12 days of inoculation.Animals were separated into groups with identical mean tumor volumes inthe range of 150-250 mm³. Treatment with the Wnt antagonist started 1-2days after grouping and the mice were dosed intraperitoneally (IP) orintravenously (IV) with 100-200 μl of Wnt antagonist, negative controlprotein CD4-Fc, or PBS negative control once daily. Subsequent drugtreatments were repeated 2-3 times weekly and continued for 3-4 weeks.Tumor volume was measured twice weekly the animals were sacrificed whenthe tumor volume reached 2500 mm³. Blood was collected during the studyby an orbital vein bleed and the serum assayed for levels of therapeuticagent by SDS-PAGE followed by immunoblot and detection using HRP orfluorescent conjugated anti-human Fc, and for activity of thetherapeutic agent by its ability to inhibit Wnt3a activation of TOPglowactivity as described in Example 7.

Allograft Tumors Inhibitory Effect of Wnt Antagonists on Growth of TumorAllografts

Treatment with Frz8-Fc by either the i.p. or i.v. routed resulted inrapid tumor regression with sustained inhibition during the course oftreatment, whereas the negative control protein CD4-Fc had no effectrelative to the PBS treatment. The treated mice were monitored for threeweeks after termination of treatment and regrowth of tumors waseventually observed.

FIG. 22 illustrates the efficacy of Frz8-Fc against MMTV-Wnt tumortransplants in athymic nude mice by intraperitoneal (IP) dosing. FIG.22A is a graph showing that nude mice hosting MMTV-Wnt-1 tumortransplants were administered PBS, CD4-Fc (10 mg/kg/day) or Frz8-Fc (10mg/kg/day) by intraperitoneal injection twice weekly. Each group had 11mice and the average tumor volume for the group was 226 mm³ before thestart of treatments. Mean tumor volume is plotted over time and thetreatment days are indicated by arrows on the X-axis. On day 25, thecontrol groups were sacrificed and the drug administration to thetreatment group stopped. FIG. 22B is tabular summary of mean tumorvolume and mean % change in tumor volume over time in the four treatmentgroups. Note that in FIG. 22B, the mean tumor volume after treatmentwith Frz8-Fc antagonist results in a reduction in tumor volume from 226mm to about 219 mm³ on the fifth day after start of treatment, and about67 mm³ on the 18^(th) day. This represents a 4% and 70%, respectively,reduction in tumor size. In this study, tumors administered the Frz-Fcantagonist showed regression in tumor size compared with controlanimals. This demonstrates that Frz-Fc antagonists of the invention aretumoricidal as a single agent and are useful as anti-cancertherapeutics.

FIG. 23 illustrates the efficacy of Frz8-Fc against MMTV-Wnt tumortransplant in athymic nude mice by intravenous (IV) dosing. FIG. 23A isa graph showing that nude mice hosting MMTV-Wnt-1 tumor transplants wereadministered PBS, CD4-Fc (10 mg/kg/day) or Frz8-Fc (10 mg/kg/day) byintravenous injection three times weekly. Each group had 11 mice and theaverage tumor volume for the group was 226 mm³ before the start oftreatments. The fourth group (high bar) in this study included 10 micewith a mean tumor volume of 375 mm³ at the start of the study that weretreated with Frz8-Fc (10 mg/kg/day) by intravenous injection three timesweekly. Mean tumor volume is plotted over time and the treatment daysare indicated by arrows on the X-axis. On day 25, the control groupanimals were sacrificed and drug administration to the treatment groupstopped. FIG. 23B is a tabular summary of mean tumor volume and mean %change in tumor volume over time in the four treatment groups. Note thatin all mice treated with Frz-Fc that the tumor burden was reduced froman average of 226 mm³ to an average volume of 179 mm³ on the 4^(th) dayafter start of treatment, and to 73 mm³ after the 18^(th) day. Thisrepresents a 21% and 67% reduction, respectively, in tumor volume. Forthe high bar group, tumor volume was reduced from an average of 376 mm³to 225 mm³ on the 4^(th) day of treatment, and to 53 mm³ on the 18^(th)day. This represents a 39% and 86% reduction, respectively, in tumorvolume.

Inhibitory Effect of Serum Obtained from Treated Mice on Wnt Signaling

Inhibition of Wnt signaling from serum isolated from the treated mice isreported in FIG. 24, with FIG. 24A showing the results of serum isolatedfrom IP treated mice, while the IV treated ones appear in FIG. 24B. Thedata is presented as a bar graph showing the Wnt signaling antagonistactivity in the TOPglow assay (as described in Example 7). The samplesappear in groups according to treatment, mouse study number anddilution. The relative luciferase activity in the TOPglow gene reporterassay is shown on the Y-axis. All samples are treated with ˜40 ng/mlpurified Wnt3a except for NA (control). All other protein controls arepresent in the medium at 5 μg/ml.

Human Xenograft Tumors

Inhibition of naturally derived human tumor models by the Wntantagonists would serve as a further indicator of their usefulness intreating human cancer. Human tumor-derived cell lines were tested forevidence of autocrine wnt signaling, similar to that seen in the PA-1teratoma cell line, as an indication of usefulness in testing Wntantagonist activity. The teratoma-derived NTera-2, Tera-2, and NCCITcell lines exhibited basal Wnt signaling that could be inhibited byFrz8-Fc, in contrast with 293 cells that exhibited low basal signalingthat was not inhibited by Frz8-Fc (FIG. 25A). Nevertheless, all fourteratoma cell lines seemed to express Wnt receptors, as signaling wasfurther stimulated by Wnt3a treatment, which could be blocked by Frz8-Fc(FIG. 25B). These results indicate that the teratoma cell lines expressWnt(s), which might contribute to their tumorigenicity. These lines weretherefore evaluated for tumor formation in athymic nude mice and basedon consistency of tumor formation, NTera-2 and PA-1 were selected for invivo efficacy studies.

Inhibitory Effect of Wnt Antagonists on Growth of NTera2 TumorXenografts

Treatment of mice exhibiting NTera2 tumor xenografts with the Wntantagonist Frz8-Fc resulted in a reduction of tumor volume byapproximately 50% and reduction tumor mass by approximately 70%,relative to the control mice.

FIG. 26 shows the anti-tumor efficacy of Frz8-Fc treatment on the growthof NTera2 tumor xenografts in athymic nude mice. Athymic nude micebearing NTera2 tumor xenografts were administered an initial dose ofPBS, CD4-Fc and Frz8-Fc at 15 mg/kg/day, followed by subsequent doses of10 mg/kg/day by intraperitoneal injection three times weekly. Each grouphad 20 mice and the average tumor volume for the group was 200 mm³before the start of treatments. The fourth group of the study included10 mice with a mean tumor volume of 336 mm³ at the start of the studythat were treated with Frz8-Fc (10 mg/kg/day) by intraperitonealinjection three times weekly. FIG. 26A is an exemplary procedural flowchart, while FIG. 26B is a graph plotting mean tumor volume over time,wherein the treatment days are indicated by arrows on the X-axis. FIG.26C is a bar graph plotting the mean tumor weights at sacrifice of allanimals in the group at day 20 of the study. FIGS. 26D and 26E aretabular summaries of mean tumor volume and mean % change in tumorvolume, respectively.

Inhibitory Effect of Serum Obtained from Mice with NTera2 TumorXenografts on Wnt Signaling

FIG. 27 is a bar graph showing Wnt signaling antagonist activity in theTOPglow assay of the Frz8-Fc Wnt antagonist of serum isolated fromvarious animals in the NTera2 tumor study. Relative luciferase activity(Y-axis) as measured from TOPglow assay from the controls and Frz8-FcWnt antagonist. No additional purified Wnt or Wnt conditioned media wasadded to the cells. These results demonstrate that reduced Wnt signalingis associated with reduction in tumor size in these mice treated withFrz8-Fc Wnt antagonist.

Inhibitory Effect of Wnt Antagonists on Growth of PA-1 Tumor Xenografts

Treatment of mice exhibiting PA-1 tumor xenografts with the Wntantagonist Frz8-Fc resulted in a significant reduction in tumor growthwithin 12 days of treatment. In this model, the tumors wereapproximately 50% smaller, with significantly smaller mass than tumorsin the control mice at the end of the treatment period.

FIG. 28 demonstrates the anti-tumor efficacy of Frz8-Fc treatment on thegrowth of PA-1 tumor xenografts in athymic nude mice. Athymic nude micebearing PA-1 tumors xenografts were administered PBS, CD4-Fc or Frz-Fcat 15 mg/kg/day, followed by subsequent doses of 10 mg/kg/day byintraperitoneal injection three times weekly. Each group had 13 mice andthe average tumor volume for the group was 168 mm³ before the start oftreatments. FIG. 28A is an exemplary procedural flow chart, while FIG.28B is a graph plotting mean tumor volume over time, wherein thetreatment days are indicated by arrows on the X-axis. FIG. 28C is agraph of mean tumor weight at sacrifice. The mice were sacrificed on day58 after cell inoculation (day 32 after start of treatments) and tumorswere excised and weighed. The mean tumor weight±SEM is plotted as afunction of the group. FIGS. 28D and 28E are tabular summaries of meantumor volume and mean % change in tumor volume, respectively.

Example 11 Wnt Signaling in Mice Transplanted with MMTV Tumors andTreated with Frz8-Fc and Frz5-Fc Wnt Antagonists Effect of Frz8-Fc andFrz5-Fc Wnt Antagonists on Wnt Signaling

Frz5-Fz inhibits Wnt3a induced signaling as effectively as Frz8-Fc.

Athymic nude mice with MMTV tumors (approximately 400-800 cubicmillimeters in size) were treated with Frz8-Fc, Frz5-Fz, or CD4-Fc, as anegative control, at 10 mg/kg. Five hours after treatment, serum wascollected by cardiac puncture from the mice and analyzed for Wntinhibiting effect on 293 cells activated with Wnt3a and transfected withTOPglow as described in Example 7. All samples are treated with ˜40ng/ml purified Wnt3a except for NA (control). All other protein controlsare present in the medium at 5 μg/ml. FIG. 29 shows the level ofinhibition in mice treated with Frz8-Fc or Frz5-Fz. Treatment withFrz8-Fc or Frz5-Fz resulted in similar levels of inhibition of Wnt 3ainduced signaling.

Effect of Frz8-Fc and Frz5-Fc Wnt Antagonists on Axin2 Expression

Frz8-Fc and Frz5-Fz compounds inhibit in vivo Wnt signaling asdetermined by modulation of the Wnt target gene Axin2

Athymic nude mice with MMTV tumors (approximately 400-800 cubicmillimeters in size) were treated with Frz8-Fc, Frz5-Fz, or CD4-Fc, as anegative control, at 10 mg/kg. Five hours after treatment, serum wascollected by cardiac puncture from the mice. RNA was extracted from thetumor cells using the QIAGEN RNAEASY kit (Qiagen, Valencia, Calif.) andanalyzed for expression of Axin2 as described in Example 9. Reducedlevels of Axin2 was observed in samples obtained from mice treated withFrz8-Fc or Frz5-Fz indicating that these compounds are able to inhibitin vivo Wnt signaling. FIG. 30 shows the reduced Axin2 expression inFrz8-Fc and Frz5-Fz treated tumor with FIG. 30A showing expressionnormalized to expression of GAPDH and FIG. 30B showing expressionnormalized to expression of rp119.

Example 12 Regenerative Tissue Treated with Wnt Antagonist

Wnt signaling plays a critical role in self-renewal of regeneratingtissue such as skin, intestine, and hematopoietic cells, and inhibitionof Wnt signaling by Dkk1 can adversely affect the architecture of thesetissues in adult mice. The following Example examines whether exposureto Frz8-Fc under the same conditions used to obtain antitumor efficacyhad any effect on intestine and skin in the mice. Tissues were collectedfrom mice that were treated in the MMTV-Wnt1 tumor model (described inExample 10) after 14 treatments, thrice a week, and sections werestained for β-catenin protein by immunohistochemistry. Analysis of skinand various intestinal compartments revealed that the architecture ofthese tissues appeared morphologically normal in treated mice of allgroups, with typical patterns of cytoplasmic and nuclear β-cateninstaining in intestinal Paneth cells (FIG. 31A) and skin hair follicles(FIG. 31B). Furthermore, histologic and immunohistochemical analysis ofskin and intestine collected from animals using the NTera-2 model, afternine treatments, thrice a week also revealed no differences betweencontrol and treated groups. This suggests that treatment with Frz8-Fcwith the therapeutic regimen that can inhibit tumor growth does not haveadverse effects on tissue renewal of skin and intestine.

Example 13

This Example describes various methods of producing the Wnt antagonists.

Expression of Wnt Antagonist in E. coli

This example illustrates preparation of an unglycosylated form of Wntantagonist by recombinant expression in E. coli.

The DNA sequence encoding Wnt antagonist is initially amplified usingselected PCR primers. The primers should contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector. A variety of expression vectors may be employed. Anexample of a suitable vector is pBR322 (derived from E. coli; seeBolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillinand tetracycline resistance. The vector is digested with restrictionenzyme and dephosphorylated. The PCR amplified sequences are thenligated into the vector. The vector will preferably include sequenceswhich encode for an antibiotic resistance gene, a trp promoter, apolyhis leader (including the first six STII codons, polyhis sequence,and enterokinase cleavage site), the Wnt antagonist coding region,lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized Wnt antagonist protein can then be purified using ametal chelating column under conditions that allow tight binding of theprotein.

Wnt antagonist may be expressed in E. coli in a poly-His tagged form,using the following procedure. The DNA encoding Wnt antagonist isinitially amplified using selected PCR primers. The primers will containrestriction enzyme sites which correspond to the restriction enzymesites on the selected expression vector, and other useful sequencesproviding for efficient and reliable translation initiation, rapidpurification on a metal chelation column, and proteolytic removal withenterokinase. The PCR-amplified, poly-His tagged sequences are thenligated into an expression vector, which is used to transform an E. colihost based on strain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts)clpP(laclq). Transformants are first grown in LB containing 50 mg/mlcarbenicillin at 30° C. with shaking until an O.D.600 of 3-5 is reached.Cultures are then diluted 50-100 fold into CRAP media (prepared bymixing 3.57 g (NH₄)₂SO₄, 0.71 g sodium citrate.2H2O, 1.07 g KCl, 5.36 gDifco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as wellas 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grownfor approximately 20-30 hours at 30° C. with shaking. Samples areremoved to verify expression by SDS-PAGE analysis, and the bulk cultureis centrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

The proteins are refolded by diluting the sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes are chosen so that the final protein concentration is between 50to 100 micrograms/ml. The refolding solution is stirred gently at 4° C.for 12-36 hours. The refolding reaction is quenched by the addition ofTFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution is filtered through a0.22 micron filter and acetonitrile is added to 2-10% finalconcentration. The refolded protein is chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance are analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein are pooled. Generally,the properly refolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded Wnt antagonist polypeptide arepooled and the acetonitrile removed using a gentle stream of nitrogendirected at the solution. Proteins are formulated into 20 mM Hepes, pH6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gelfiltration using G25 Superfine (Pharmacia) resins equilibrated in theformulation buffer and sterile filtered.

Expression of Wnt Antagonist in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof Wnt antagonist by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the Wnt antagonist DNA is ligatedinto pRK5 with selected restriction enzymes to allow insertion of theWnt antagonist DNA using ligation methods such as described in Sambrooket al., supra. For purposes of this example, the resulting vector isreferred to as pRK5-WA.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μg pRK5-WADNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappayaet al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl,0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added, dropwise, 500 μlof 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄, and a precipitateis allowed to form for 10 minutes at 25° C. The precipitate is suspendedand added to the 293 cells and allowed to settle for about four hours at37° C. The culture medium is aspirated off and 2 ml of 20% glycerol inPBS is added for 30 seconds. The 293 cells are then washed with serumfree medium, fresh medium is added and the cells are incubated for about5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml³⁵S-cysteine and 200 μCi/ml³⁵S-methionine. After a12 hour incubation, the conditioned medium is collected, concentrated ona spin filter, and loaded onto a 15% SDS gel. The processed gel may bedried and exposed to film for a selected period of time to reveal thepresence of Wnt antagonist polypeptide. The cultures containingtransfected cells may undergo further incubation (in serum free medium)and the medium is tested in selected bioassays.

In an alternative technique, Wnt antagonist may be introduced into 293cells transiently using the dextran sulfate method described bySomparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells aregrown to maximal density in a spinner flask and 700 μg pRK5-WA DNA isadded. The cells are first concentrated from the spinner flask bycentrifugation and washed with PBS. The DNA-dextran precipitate isincubated on the cell pellet for four hours. The cells are treated with20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing expressed Wnt antagonist can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

In another embodiment, Wnt antagonist can be expressed in CHO cells. ThepRK5-WA can be transfected into CHO cells using known reagents such asCaPO₄ or DEAE-dextran. As described above, the cell cultures can beincubated, and the medium replaced with culture medium (alone) or mediumcontaining a radiolabel such as ³⁵S-methionine. After determining thepresence of Wnt antagonist polypeptide, the culture medium may bereplaced with serum free medium. Preferably, the cultures are incubatedfor about 6 days, and then the conditioned medium is harvested. Themedium containing the expressed Wnt antagonist can then be concentratedand purified by any selected method.

Epitope-tagged Wnt antagonist may also be expressed in host CHO cells.The Wnt antagonist may be subcloned out of the pRK5 vector. The subcloneinsert can undergo PCR to fuse in frame with a selected epitope tag suchas a poly-his tag into a Baculovirus expression vector. The poly-histagged Wnt antagonist insert can then be subcloned into a SV40 drivenvector containing a selection marker such as DHFR for selection ofstable clones. Finally, the CHO cells can be transfected (as describedabove) with the SV40 driven vector. Labeling may be performed, asdescribed above, to verify expression. The culture medium containing theexpressed poly-His tagged Wnt antagonist can then be concentrated andpurified by any selected method, such as by Ni′-chelate affinitychromatography.

Wnt antagonist may also be expressed in CHO and/or COS cells by atransient expression procedure or in CHO cells by another stableexpression procedure.

Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used expression in CHO cellsis as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents SUPERFECTt® (Quiagen), DOSPER® or FUGENE®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁷ cells are frozen in an ampule for furthergrowth and production as described below.

The ampules containing the plasmid DNA are thawed by placement intowater bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3 L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH is determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins are purified using aNi-NTA column (Qiagen). Before purification, imidazole is added to theconditioned media to a concentration of 5 mM. The conditioned media ispumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5ml/min. at 4° C. After loading, the column is washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein is subsequentlydesalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Expression of Wnt Antagonist in Yeast

The following method describes recombinant expression of Wnt antagonistin yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of Wnt antagonist from the ADH2/GAPDH promoter.DNA encoding Wnt antagonist and the promoter is inserted into suitablerestriction enzyme sites in the selected plasmid to direct intracellularexpression of Wnt antagonist. For secretion, DNA encoding Wnt antagonistcan be cloned into the selected plasmid, together with DNA encoding theADH2/GAPDH promoter, a native Wnt antagonist signal peptide or othermammalian signal peptide, or, for example, a yeast alpha-factor orinvertase secretory signal/leader sequence, and linker sequences (ifneeded) for expression of Wnt antagonist.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant Wnt antagonist can subsequently be isolated and purified byremoving the yeast cells from the fermentation medium by centrifugationand then concentrating the medium using selected cartridge filters. Theconcentrate containing Wnt antagonist may further be purified usingselected column chromatography resins.

Expression of Wnt Antagonist in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of Wnt antagonistin Baculovirus-infected insect cells.

The sequence coding for Wnt antagonist is fused upstream of an epitopetag contained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding Wnt antagonist or the desired portion of the codingsequence of Wnt antagonist such as the sequence encoding anextracellular domain of a transmembrane protein or the sequence encodingthe mature protein if the protein is extracellular is amplified by PCRwith primers complementary to the 5′ and 3′ regions. The 5′ primer mayincorporate flanking (selected) restriction enzyme sites. The product isthen digested with those selected restriction enzymes and subcloned intothe expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BACULOGOLD™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged Wnt antagonist can then be purified, forexample, by Ni²⁺-chelate affinity chromatography as follows. Extractsare prepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or Western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged Wnt antagonist are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) Wntantagonist can be performed using known chromatography techniques,including for instance, Protein A or protein G column chromatography.

Purification of Wnt Antagonist Polypeptides Using AffinityChromatography

Native or recombinant Wnt Antagonist polypeptides may be purified by avariety of standard techniques in the art of protein purification. Forexample, pro-, mature, or pre-Wnt antagonist polypeptide is purified byimmunoaffinity chromatography using antibodies specific for the Wntantagonist polypeptide of interest. In general, an immunoaffinity columnis constructed by covalently coupling the Wnt antagonist polypeptide toan activated chromatographic resin. Alternatively, Wnt antagonist whichcontain an Fc domain may be purified directly from media using aimmobilized protein A resin such as ProSepA (Millipore).

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column may be utilized in the purification of Wntantagonist polypeptide by preparing a fraction from cells containing Wntantagonist in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble Wnt antagonistpolypeptide containing a signal sequence may be secreted in usefulquantity into the medium in which the cells are grown.

A soluble Wnt antagonist polypeptide-containing preparation is passedover the immunoaffinity column, and the column is washed underconditions that allow the preferential absorbance of Wnt antagonistpolypeptide (e.g., high ionic strength buffers in the presence ofdetergent). Then, the column is eluted under conditions that disruptantibody/Wnt antagonist binding (e.g., a low pH buffer such asapproximately pH 2-3, or a high concentration of a chaotrope such asurea or thiocyanate ion), and Wnt antagonist polypeptide is collected.

BIBLIOGRAPHY

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1-32. (canceled)
 33. A soluble receptor comprising (a) a fragment of anextracellular domain of a human Frizzled (Frz) receptor and (b) a humanFc domain, wherein the fragment of the extracellular domain of the humanFrz receptor consists essentially of an amino acid sequence selectedfrom the group consisting of amino acid residues 1 to 156 of SEQ IDNO:42, amino acid residues 1 to 129 of SEQ ID NO:25, amino acid residues1 to 155 of SEQ ID NO:39; and amino acid residues 1 to 129 of SEQ IDNO:22, wherein the soluble receptor has a longer half-life in vivo thana soluble receptor comprising the extracellular domain of the Frzreceptor and the human Fc domain.
 34. The soluble receptor of claim 33wherein the human Fc is human IgG1 Fc comprising the amino acid sequenceof SEQ ID NO:67.
 35. A pharmaceutical composition comprising the solublereceptor of claim
 33. 36. A kit comprising the soluble receptor of claim33.
 37. The soluble receptor of claim 33, wherein the soluble receptorinhibits the Wnt-dependant growth of solid tumor cells.
 38. The solublereceptor of claim 33, wherein the soluble receptor inhibits theWnt-dependant growth of breast tumor cells.
 39. An isolated polypeptidecomprising an amino acid sequence having at least 95% sequence identityto an amino acid sequence selected from the group consisting of aminoacid residues 1 to 129 of SEQ ID NO:25 and amino acid residues 1 to 129of SEQ ID NO:22, wherein said polypeptide is a soluble receptor thatinhibits the Wnt-dependent growth of solid tumor cells.
 40. A solublereceptor comprising (a) a fragment of an extracellular domain of a humanFrizzled (Frz) receptor and (b) a human Fc domain, wherein the fragmentof the extracellular domain of the human Frz receptor consistsessentially of an amino acid sequence selected from the group consistingof amino acid residues 1 to 156 of SEQ ID NO:42 and amino acid residues1 to 155 of SEQ ID NO:39, and wherein the soluble receptor has ahalf-life in vivo of at least 24 hours in mice following i.p. injection.41. A soluble receptor comprising (a) a fragment of an extracellulardomain of a human Frizzled (Frz) receptor and (b) a human Fc domain,wherein the fragment of the extracellular domain of the human Frzreceptor consists essentially of an amino acid sequence selected fromthe group consisting of amino acid residues 1 to 156 of SEQ ID NO:42 andamino acid residues 1 to 155 of SEQ ID NO:39, and wherein the solublereceptor is detectable in serum at least 24 hours following i.p.injection in mice.
 42. A method of inhibiting the growth of solid tumorcells in a subject in need thereof, the method comprising administeringto the subject the soluble receptor of claim 33 in an amount effectiveto inhibit the Wnt-dependent growth of solid tumor cells.
 43. A methodof inhibiting the growth of solid tumor cells in a subject in needthereof, the method comprising administering to the subject the solublereceptor of claim 40 in an effective amount to inhibit the Wnt-dependentgrowth of solid tumor cells.
 44. A method of inhibiting the growth ofsolid tumor cells in a subject in need thereof, the method comprisingadministering to the subject the soluble receptor of claim 41 in aneffective amount to inhibit the Wnt-dependent growth of solid tumorcells.
 45. The method of claim 42, wherein the soluble receptor isadministered with radiation therapy.
 46. The method of claim 42, whereinthe soluble receptor is administered with chemotherapy.
 47. The methodof claim 42, wherein the solid tumor cells are from a breast tumor,colorectal tumor, lung tumor, pancreatic tumor, prostate tumor, or ahead and neck tumor.